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Histochemical study of lectin binding in the major salivary glands of adult fallow-deer (Dama dama L.)
Acta histochem. 99, 81-89 (1997) Gustav Fischer Verlag
Aela hisloeMMiea
Histochemical study of lectin binding in the major salivary glands of adult fallow-deer (Dama dama L.) Vera Pedinil, Piero Ceccarelli 2 , Anna Maria Gargiulo l and Cecilia Dall'Agliol I Department of Anatomy for Domestic Ammals. Faculty of Veterinary Medicine. Via S. Costanzo 4. I-06l26 Perugla. Italy and 2 Faculty of VeterInary MedIcIne. Via R. Fidanza 5. 1-62024 Matehca. Italy Accepted 27 October 1996
Summary
The sugar residues in glycoconjugates present in the parotid and mandibular glands of the adult fallow-deer were detected and characterized by using a battery of eight different lectin-horseradish peroxidase conjugates. In some cases a treatment with sialidase preceded the lectin staining. Parotid secretory cells produced glycoconjugates with N-acetylgalactosamine, N-acetylglucosamine and mannose residues. Mucous acinar cells were the most reactive sites of the mandibular gland and contained conspicuous quantities of oligosaccharides with terminal sialic acid radicals. Galactosil-(fJl ~ 3)N-acetylgalactosamine was the most abundant penultimate sugar linked to N-acetylneuraminic acid. Mandibular mucous cells also presented N-acetylglucosamine and sialylated components with the terminal dimer sialic acid-N-acetylgalactosamine. Demilunar cells contained glycoconjugates with fucose and mannose residues. The apical surface of duct cells was stained by all the lectins. Key words: salivary glands - fallow-deer - lectins - histochemistry
Introduction
Salivary glycoproteins consist of a high number and an extreme diversity of carbohydrate side chains attached to a polypeptide backbone (Nieuw Amerongen et aI., 1995; Tabak, 1995). Many studies have demonstrated the importance of glycosidic residues for the structural and functional properties of glycoproteins, and the advent of lectin histochemistry has allowed carbohydrate moieties to be characterized in situ (Damianov, 1987; Spicer and Schulte, 1992). This method has widely been used to study stored secretory glycoproteins in the major salivary glands of many mammals (Menghi and Materazzi. 1994). The major salivary glands of the fallow-deer have been the subject of a topographic study (Saber and Hofmann, 1984) and more recently of an investigation which used traditional histochemical procedures (Pedini et aI., 1995). The aim of the present study was to investigate the distribution of sugar residues in the parotid and Correspondence to. V. Pedim
82
V. Pedini et a1.
mandibular glands of the adult fallow-deer by using nine horseradish peroxidase-conjugate lectins, combined with enzymatic treatment. Materials and Methods Tissue preparatIOn. Five clinically healthy adult fallow-deers of both sexes, killed at the slaughterhouse of "Cooperativa Rinascita Montana", were used. Some specimens of the mandibular and parotid glands were obtained Immediately after killing and 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-HgCh were treated with Lugol's solutIon prior to staining. Lectin histochemistry. Sections were incubated in a 10-4011glml of lectin-horseradish peroxidase conjugate (Sigma. St. Louis, USA) solution in 0.1 M phosphate-buffered sahne (PBS). pH 7.2 containing 0.1 mM CaCI 2 • MnCl 2 and MgCI2 for 1 h at room temperature. After washing in phosphate-buffered saline. lectin binding sites were revealed using a diaminobenzidine-hydrogen peroxide medium for IS min at room temperature (Schulte et al.. 1985). The horseradish peroxidase-lectin conjugates used and their sugar specificities are listed in Table 1.
Table 1. Lectins used for the histochemical studies on the fallow-deer parotid gland Source of lectin
Abbreviation
Carbohydrate binding specificity
Arachis hypogea Ricinus communis Dolichos biflorus Glycine max Triticum vulgarIS Lotus tetragonolobus Ulex europaeus Canavalia enslformls
PNA RCA I DBA SBA
gal-(p1 ~ 3)galNAc p-galactose a- N -acetylgalactosamine a,p-N-acetylgalactosamine N-acetylglucosamine a-fucose a-fucose D-mannose > D-glucose
gal-(pl
~
WGA
LTA UEAI ConA
3)galNAc = galactosyl-(P1
~
3)N-acetylgalactosamine.
For the lectin labelling controls. sections were incubated in lectin solutions to which 0.2 M hapten sugars were added. Enzyme digestion. Enzymatic treatment was carned out on adjacent sections. Prior to lectin histochemistry, some sections were incubated at 37°C for 16 h in a 0.8 IUlml solution of neuraminidase (Neu) (siaJidase) from Clostridium perfrigens in 0.1 M sodium acetate buffer. pH 5.5 containing 10 mM CaCl2 . Removal of siahc acid was confirmed on adjacent sections by the lack of staining With AJcian blue at pH 2.5. The controls for sialidase digestion were also exposed to the buffer in which the enzyme was dissolved. Sialic acid residues with O-acetyl substituents at C-4 resisted sialidase treatment (Moschera and Pigman, 1975) but were cleaved after the removal of acetyl groups by saponification which was performed by immersing the sections in a 1 % solution of potassium hydroxide III 70% ethanol for 15 min at room temperature.
Results The parotid and mandibular glands of the fallow-deer presented the classic morphology of the salivary glands. There were typical tubuloacinar glands, and the parenchymal components consisted of acinar endpieces, intercalated ducts, striated ducts and excretory ducts. The parotid glands had serous acini while the mandibular glands were characterized by mucous acini surrounded by serous demilunar cells. The distribution and intensity of the most representative lectin binding sites in parotid and mandibular glands of fallow-deer are summarized in Table 2.
Lectin histochemistry in fallow-deer salivary glands
83
Table 2. Lectin binding In the salivary glands of the fallow-deer
Parotid gland Acmar cells Stnated duct Apical surface Cytoplasm Mandibular gland Acinar cells Demilunar cells Stnated duct Apical surface Cytoplasm
PNA
NEU- RCA I DBA PNA
0
0
0
3 0-2
3 0-2
3 0-2
0
1 0
4 0
0
1 0
0
3 0
2 0
()
NEU- SBA WGA NEU- LTA DBA WGA
UEA I ConA
3
3
3
0
3*
3
0
3 0
3 0
3 0
2 0-2
3 0-3
2 2
2 0
2 0
2 0
2 0
0 1-3
0
1-3
0 2
3
2 2
3 0
3
2 0
4 0-3
2 2
0
Numbers indicate stammg intensity on a subjectively estlmated scale from 0, unreactive, to 4, most reactive * UEA I staimng was locahzed m the apical cytoplasm and at the surface.
Parotid gland. Secretory cells reacted intensely with SBA (Fig. I), ConA (Fig. 2) and WGA; the reaction product was distributed throughout the cytoplasm. Sialidase treatment did not affect WGA positivity. Acinar cells showed a characteristic staining with UEA I (Fig. 3), localized only in the apical cytoplasm and on the surface. DBA (Fig. 4) produced a weak reaction in secretory cells; this result was unaffected by neuraminidase digestion. DBA was also bound to vascular endothelial cells in vessels of every caliber. Parotid secretory cells did not react with PNA, Neu-PNA, RCA I and LTA. Saponification before enzymatic digestion did not affect the PNA and DBA results. The apical surface of striated duct cells bound all the lectin conjugates with a variable avidity. ConA (Fig. 2) also stained the cytoplasm of striated duct cells; the reaction product was uniform and distibuted in all the cells. UEA I (Fig. 3), LTA, RCA I and PNA marked the cytoplasm of the cells lining striated ducts with a variable intensity ranging from negative to strong staining. The luminal surface of interlobular duct cells reacted with all lectins employed. Cells resembling mucous goblets, localized in the interlobular ducts, reacted intensely with PNA (Fig. 5), WGA. UEA L LTA and less heavily with the other lectins. These reactive cells increased in number in the distal direction making up about 3040% of the epithelial cells lining the larger interlobular ducts. Mandiblliar gland. SBA and WGA (Fig. 6) stained acinar cells with moderate intensity, and WGA positivity was unaffected by neuraminidase digestion. The sequence Neu-PNA (Fig. 8) caused the strongest reaction in the acinar cells. PNA without neuraminidase digestion (Fig. 7) also produced positive staining but of less intensity. Acinar cells were faintly reactive with DBA (Fig. 9) and showed a stronger positivity when the lectin reaction was preceded by enzymatic treatment (Fig. 10). As in the parotid gland, the vascular endothelium reacted with DBA as well. Potassium hydroxide treatment prior to sialidase degradation did not affect the PNA and DBA reactivity. LTA, UEA I and ConA did not mark mandibular acinar cells. Demilunar cells only reacted with Con A, LTA and UEA I. The staining obtained after ConA incubation was homogeneous and discrete (Fig. 11), while LTA and UEA I (Fig. 12) yielded variable positivity in the demilunar cells, ranging from negative to moderate staining.
V. Pedini et al.
84
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Fig. 1. Parotid gland, SBA. All acinar cells are heavily stained as well as the apical surface of striated re activity is present in secretory duct cells (arrows). x350. Fig. 2. Parotid Parotid gland, ConA. An intense reactivity cells. The cytoplasm of cells linmg striated ducts shows uniform staining. x 350. Fig. 3. Parotid gland, UEA 1. Acinar cells with positive reaction localized in the apical cytoplasm and at the surface. The apex of all striated duct cells shows an intense affinity for this lectin while the cytoplasm exhibits light to heavy staining. x350. Fig. 4. Parotid gland, DBA. Acinar cells stain weakly. Note the endothelium staining of the capillaries (arrows) and a vessel (arrowhead). x 380.
Lectin histochemistry in fallow-deer salivary glands
85
Fig. 5. Parotld gland, PNA Apices of cells hning the interlobular duct react with PNA. Some cells also show an intense cytoplasmic positiVity. x 300. Fig. 6. Mandibular gland. WGA. Acinar cells exhibit a moderate staining; demllunes are unreactive (asterisks). The luminal surface of striated duct cells stains intensely (arrow). x350. Fig. 7. Mandibular gland, PNA. A weak staming IS locahzed m the aCInar c ells and apex of cells lining a stnated duct (arrow). Demilunar cells are unstained (asterisks). x 410. Fig. 8. Mandibular gland. Neu-PNA. Siahdase digestion enhance acinar cell reactivity and apIcal positivity of duct cells (arrow) for PNA while demilunes remain unreactive (asterisks). x410
V. Pedini et al.
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Fig. Fig. 9. 9. Mandibular gland, DBA. Acinar cells and apices of striated duct cells stain weakly; demilunes are unstained. Vascular endothelial cells react with DBA (arrows). xx410. 410. Fig. 10. Mandibular gland, Neu·DBA. Sialidase digestion promotes increased DBA reactivity in the acinar cells and at the apical Neu-DBA. surface of striated duct cells (arrow). A weak cytoplasmic positivity also appears in these last cells. Demilunes remain unreactive (asterisks). x41O. Fig. 11. Mandibular gland, ConA. Demilunar cells and cells lining striated ducts show an uniform staining throughout the cytoplasm. Acinar cells are unstained. x 350. Fig. 12. Mandibular gland, UEA I. Demilunar cells exhibit a variable staining with UEA I while acinar cells are unreactive. unreactive. Striated duct cells show an intense posItivity at the luminal surface. Some cells present also staining in the supranuclear portion (arrows). (arrows). x350
Lectin histochemistry in fallow-deer salivary glands
87
The apical surface of cells lining all the different segments of the excretory system presented a lectin staining pattern similar to that obtained in the parotid gland. The only difference was the intensification of PNA and DBA reactivity after neuraminidase digestion. In addition to the staining of the luminal surface, striated duct cells showed uniform staining throughout the cytoplasm with the NEU-DBA sequence (Fig. 10), ConA (Fig. 11) and SBA. UEA I caused an intense staining in a few cells lining striated ducts; the reaction product was localized in the supranuclear cytoplasm (Fig. 12). Controls. All staining by the lectins was completely inhibited when the lectin-peroxidase conjugates were incubated with the appropriate specific sugar.
Discussion Parotid and mandibular glands of the fallow-deer are structurally and topographically similar to those of most ruminant species (Saber and Hofmann, 1984). Our previous histochemical investigations of the major salivary glands of fallow-deers (Pedini et aI., 1995) demonstrated that the acinar cells of the mandibular gland mainly possess acid glycoconjugates with sialic acid and carboxyls, while parotid secretory cells and mandibular demilunar cells have neutral glycoproteins in their secretion. These studies also demonstrated the absence of neuraminic acid in parotid secretory cells of the fallow-deer. The lectin histochemistry results reported in the present study confirm these data. The uniform and intense SBA staining in parotid secretory cells of the fallowdeer could be attributed to the homogeneous presence of glycoconjugates containing N-acetylgalactosamine. This sugar residue is particularly present in the p-anomeric form as demonstrated by the weaker result obtained with DBA, a lectin which shows no affinity for p-N-acetylgalactosamine (Schulte et aI., 1985). The faint DBA reactivity and PNA negativity were unaffected by neuraminidase digestion indicating the absence of terminal sialic acid linked to D-galactose-(p1 ~ 3)N-acetylgalactosamine or N-acetylgalactosamine. Parotid glands of others mammals (cat: Accili et aI., 1989; rat. rabbit and hare: Accili et aI., 1992; dog: Pedini et aI., 1994 a) contain these sialoglycoconjugates in their secretion. However. already in 1962. Shackleford and Klapper observed morphological and histochemical differences comparing ruminant parotid glands with the same organ in other mammals. Moreover. lectin histochemical studies have never been carried out on the ruminant parotid gland. The intense reaction obtained with ConA and WGA shows the presence of mannose and N-acetylglucosamine in the acinar cells of the fallow-deer parotid gland. The latter lectin also recognizes sialic acid (Monsigny et aI., 1980) but the WGA staining did not change when sialidase treatment preceded lectin incubation; the absence of neuraminic acid in parotid secretory cells of the fallow-deer has been shown in our previous study (Pedini et aI., 1995). The mucous acinar cells of the mandibular gland contain acidic glycoconjugates with terminal sialic acid residues indicated by their loss of affinity for Alcian blue after neuraminidase digestion (Pedini et al.. 1995). The increased reactivity of PNA and DBA following the enzymatic removal of sialic acid confirms this result, showing the presence of sialic acid linked to D-galactose-(p1 ~ 3)N-acetylgalactosamine and N-acetylgalactosamine, respectively. The mandibular gland of other ruminants (ovine: Schulte et aI., 1985; bovine: Menghi et aI., 1992) contains the terminal trisaccharide sialic acid-D-galactose-CP1 ~ 3)N- acetylgalactosamine and the terminal disaccharide sialic acid-N-acetylgalactosamine in the glycoconjugates secreted by the mucous cells. The failure of saponification before sialidase digestion to increase PNA
88
V. Pedini et aI.
and DBA reactivity indicate the absence or presence of only low levels of sialic acid residues with O-acetyl substituents at C4 (Schulte et aI., 1985). Mandibular acinar cells also showed a moderate affinity for WGA, unaffected by sialidase digestion, which shows the presence of N-acetylglucosamine residues. This sugar is partly present in the dimer D-galactose-(p1 ~ 4)N-acetylglucosamine as indicated by the weak positivity of RCA I. An interesting finding is the demilunar affinity for UEA I, LTA and ConA, which reflects the presence of glycoconjugates with fucose and mannose residues. Serous demilunes lack detectable complex carbohydrate in sheep mandibular gland (Schulte et aI., 1985) and react with DBA and WGA only in the bovine mandibular gland (Menghi et aI., 1992). However, Spicer and Schulte (1992) report in a recent review on cell glycoconjugates that " ... comparative studies have shown marked dissimilarities among genera and species in the glycoconjugates present at a given site. Cells exhibiting such variability have been found in salivary glands, kidney, trachea, and other tissues ..." Cells lining striated and interlobular ducts showed a predominant lectin-affinity at their apical surface, both in the parotid and in the mandibular gland. All the lectins employed produced a positivity of variable intensity at these sites. The only difference between parotid and mandibular gland is the presence of a sialoglycoconjugate in the apical plasmalemma of ductal cells, as demonstrated by the increased PNA and DBA reaction following sialidase digestion in the latter gland. Terminal sialic acid has been found at the apical surface of ductal cells in the mandibular glands of other mammals (Schulte et aI., 1985; Menghi et aI., 1992; Pedini et aI., 1994 b). The cytoplasm of striated duct cells mainly reacted with ConA and UEA I. The positivity of ConA is probably due to the presence of glucose residues in glycogen, which may represent an energy reserve for the production of hypotonic saliva by the striated ducts (Fawcett, 1986; Menghi et al., 1987). Fucosylated glycoconjugates, demonstrated by UEA I affinity, have already been shown in the cytoplasm of some cells lining striated ducts in the ovine mandibular gland (Schulte et aI., 1985). Their presence may be connected to the relative hydrophobicity of this sugar, which could contribute to the transport functions of duct cells. Endothelial cells lining the vasculature had affinity for DBA in both salivary glands examined. DBA binds to the plasma membrane of capillary endothelial cells in a variety of tissues of the mouse (Ponder, 1983), whereas UEA I is considered as a marker for human blood vessels (Damianov, 1987). Further investigations could verify the DBA positivity of endothelial cells in other organs of the fallow-deer.
Acknowledgements The authors wish to thank Mrs Gabriella Mancini for her excellent technical assistance. The authors are also grateful to the president and technical staff of the "Cooperativa Rinascita Montana di Nocera Umbra" for invaluable aid in collecting material. This work was partly supported by grants (40% and 60%) from MURST.
References Accili D, Menghi G, Bondi AM, and Scocco P (1992) Glycoconjugate compOSition of mammalian parotid glands elucidated In situ by lectins and glycosidases. Acta histochem 92: 196-206 Accili D, Menghi G, and Materazzi G (1989) Biochemical analysis and lectin histochemistry for detecting complex carbohydrates in the cat parotid gland. Arch BioI 100: 337-351
Lectin histochemistry in fallow-deer salivary glands
89
Damianov I (1987) BIOlogy of disease: lectin cytochemistry and histochemistry. Lab Invest 57: 5-18 Fawcett OW (1986) A textbook of histology. Saunders WB. Philadelphia. pp 593-597 Menghi G. Accili D. and Bondi AM (1987) Differential binding sites of peroxidase-labelled lectins In the submandibular gland of sucking and adult cats. Acta histochem 82: 63-75 Menghl G. Accili D. Scocco P. and Materazzi G (1992) Sialoglycoderivatives of bOVine submandibular gland Identified in situ by histochemical techmques combined with lectins. HistochemIstry 97: 397-403 Menghi G. and Materazzl G (1994) Exoglycosidases and lectlns as sequencing approaches of salivary gland olIgosaccharides. Histol Histopath 9: 173-183 Monsigny M. Roche AC, Sene C, Maget-Dana R. and Delmotte F (1980) Sugars-lectin interactions: how does wheat germ agglutinin bind sialoglycoconJugates. Eur J Biochem 104: 147-152 Moschera1. and Pigman W (1975) The isolation and characterization of rat sublingual mucus glycoprotein. Carbohydr Res 40' 53-67 Nleuw Amerongen AV. Bolscher JGM. and Veerman ECI (1995) Salivary mucins. protective functIons In relation to their diverSity. Glycobiol 5: 773- 740 Pedlm V. Ceccarelli P. and GargIUlo AM (1994 a) LocalizatIon of glycoconJugates in dog parotid gland by lectin histochemistry. Vet Res Comm 18: 269-279 Pedini V. CeccarellI P. and Gargiulu AM (1994 b) Glycoconjugates in the mandibular salIvary gland of adult dogs revealed by lectin histochemistry. Res Vet SCI 57: 353-357 Pedlni V. Fagioli 0, and Lorvik S (1995) Histochemical stud on the major salivary glands of fallow deer (Dama dama L.). Atti SISVet. in press Ponder BAJ (1983) Lectin histochemistry. In: Polak 1M. Van Noorden S (Eds) Immunocytochemistry: practIcal applicatIOns In pathology and biology. Wright PSG. Bristol. pp 129- 142 Saber AS. and Hofmann RR (1984) Comparallve anatomical and topographic studies of the salivary glands of red deer (Cervus elaphus). fallow deer (Cervus dama) . and mouflon (OVIS ammon ml/simon) - Ruminantia. Cervidae. Bovidae. Gegenbaurs morph lahrb 130: 273-286 Schulte BA. Spicer SS. and Miller RL (1985) Lectin histochemistry of secretory and cell-surface glycoconjugates in the ovine submandibular gland. Cell Tissue Res 240: 57-66 Shackleford JM. and Klapper CE (1962) Structure and carbohydrate histochemistry of mammalian salivary glands. Am 1 Anat 111: 25-47 Spicer SS. and Schulte BA (1992) Diversity of cell glycoconJugates shown histochemically: a perspectlve. l Hlstochem Cytochem 40: 1- 38 Tabak LA (1995) In defense of the oral cavity: structure. biosynthesis. and function of salivary muclns. Ann Rev Physiol 57: 547- 564
Aela hisloeMMiea
Histochemical study of lectin binding in the major salivary glands of adult fallow-deer (Dama dama L.) Vera Pedinil, Piero Ceccarelli 2 , Anna Maria Gargiulo l and Cecilia Dall'Agliol I Department of Anatomy for Domestic Ammals. Faculty of Veterinary Medicine. Via S. Costanzo 4. I-06l26 Perugla. Italy and 2 Faculty of VeterInary MedIcIne. Via R. Fidanza 5. 1-62024 Matehca. Italy Accepted 27 October 1996
Summary
The sugar residues in glycoconjugates present in the parotid and mandibular glands of the adult fallow-deer were detected and characterized by using a battery of eight different lectin-horseradish peroxidase conjugates. In some cases a treatment with sialidase preceded the lectin staining. Parotid secretory cells produced glycoconjugates with N-acetylgalactosamine, N-acetylglucosamine and mannose residues. Mucous acinar cells were the most reactive sites of the mandibular gland and contained conspicuous quantities of oligosaccharides with terminal sialic acid radicals. Galactosil-(fJl ~ 3)N-acetylgalactosamine was the most abundant penultimate sugar linked to N-acetylneuraminic acid. Mandibular mucous cells also presented N-acetylglucosamine and sialylated components with the terminal dimer sialic acid-N-acetylgalactosamine. Demilunar cells contained glycoconjugates with fucose and mannose residues. The apical surface of duct cells was stained by all the lectins. Key words: salivary glands - fallow-deer - lectins - histochemistry
Introduction
Salivary glycoproteins consist of a high number and an extreme diversity of carbohydrate side chains attached to a polypeptide backbone (Nieuw Amerongen et aI., 1995; Tabak, 1995). Many studies have demonstrated the importance of glycosidic residues for the structural and functional properties of glycoproteins, and the advent of lectin histochemistry has allowed carbohydrate moieties to be characterized in situ (Damianov, 1987; Spicer and Schulte, 1992). This method has widely been used to study stored secretory glycoproteins in the major salivary glands of many mammals (Menghi and Materazzi. 1994). The major salivary glands of the fallow-deer have been the subject of a topographic study (Saber and Hofmann, 1984) and more recently of an investigation which used traditional histochemical procedures (Pedini et aI., 1995). The aim of the present study was to investigate the distribution of sugar residues in the parotid and Correspondence to. V. Pedim
82
V. Pedini et a1.
mandibular glands of the adult fallow-deer by using nine horseradish peroxidase-conjugate lectins, combined with enzymatic treatment. Materials and Methods Tissue preparatIOn. Five clinically healthy adult fallow-deers of both sexes, killed at the slaughterhouse of "Cooperativa Rinascita Montana", were used. Some specimens of the mandibular and parotid glands were obtained Immediately after killing and 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-HgCh were treated with Lugol's solutIon prior to staining. Lectin histochemistry. Sections were incubated in a 10-4011glml of lectin-horseradish peroxidase conjugate (Sigma. St. Louis, USA) solution in 0.1 M phosphate-buffered sahne (PBS). pH 7.2 containing 0.1 mM CaCI 2 • MnCl 2 and MgCI2 for 1 h at room temperature. After washing in phosphate-buffered saline. lectin binding sites were revealed using a diaminobenzidine-hydrogen peroxide medium for IS min at room temperature (Schulte et al.. 1985). The horseradish peroxidase-lectin conjugates used and their sugar specificities are listed in Table 1.
Table 1. Lectins used for the histochemical studies on the fallow-deer parotid gland Source of lectin
Abbreviation
Carbohydrate binding specificity
Arachis hypogea Ricinus communis Dolichos biflorus Glycine max Triticum vulgarIS Lotus tetragonolobus Ulex europaeus Canavalia enslformls
PNA RCA I DBA SBA
gal-(p1 ~ 3)galNAc p-galactose a- N -acetylgalactosamine a,p-N-acetylgalactosamine N-acetylglucosamine a-fucose a-fucose D-mannose > D-glucose
gal-(pl
~
WGA
LTA UEAI ConA
3)galNAc = galactosyl-(P1
~
3)N-acetylgalactosamine.
For the lectin labelling controls. sections were incubated in lectin solutions to which 0.2 M hapten sugars were added. Enzyme digestion. Enzymatic treatment was carned out on adjacent sections. Prior to lectin histochemistry, some sections were incubated at 37°C for 16 h in a 0.8 IUlml solution of neuraminidase (Neu) (siaJidase) from Clostridium perfrigens in 0.1 M sodium acetate buffer. pH 5.5 containing 10 mM CaCl2 . Removal of siahc acid was confirmed on adjacent sections by the lack of staining With AJcian blue at pH 2.5. The controls for sialidase digestion were also exposed to the buffer in which the enzyme was dissolved. Sialic acid residues with O-acetyl substituents at C-4 resisted sialidase treatment (Moschera and Pigman, 1975) but were cleaved after the removal of acetyl groups by saponification which was performed by immersing the sections in a 1 % solution of potassium hydroxide III 70% ethanol for 15 min at room temperature.
Results The parotid and mandibular glands of the fallow-deer presented the classic morphology of the salivary glands. There were typical tubuloacinar glands, and the parenchymal components consisted of acinar endpieces, intercalated ducts, striated ducts and excretory ducts. The parotid glands had serous acini while the mandibular glands were characterized by mucous acini surrounded by serous demilunar cells. The distribution and intensity of the most representative lectin binding sites in parotid and mandibular glands of fallow-deer are summarized in Table 2.
Lectin histochemistry in fallow-deer salivary glands
83
Table 2. Lectin binding In the salivary glands of the fallow-deer
Parotid gland Acmar cells Stnated duct Apical surface Cytoplasm Mandibular gland Acinar cells Demilunar cells Stnated duct Apical surface Cytoplasm
PNA
NEU- RCA I DBA PNA
0
0
0
3 0-2
3 0-2
3 0-2
0
1 0
4 0
0
1 0
0
3 0
2 0
()
NEU- SBA WGA NEU- LTA DBA WGA
UEA I ConA
3
3
3
0
3*
3
0
3 0
3 0
3 0
2 0-2
3 0-3
2 2
2 0
2 0
2 0
2 0
0 1-3
0
1-3
0 2
3
2 2
3 0
3
2 0
4 0-3
2 2
0
Numbers indicate stammg intensity on a subjectively estlmated scale from 0, unreactive, to 4, most reactive * UEA I staimng was locahzed m the apical cytoplasm and at the surface.
Parotid gland. Secretory cells reacted intensely with SBA (Fig. I), ConA (Fig. 2) and WGA; the reaction product was distributed throughout the cytoplasm. Sialidase treatment did not affect WGA positivity. Acinar cells showed a characteristic staining with UEA I (Fig. 3), localized only in the apical cytoplasm and on the surface. DBA (Fig. 4) produced a weak reaction in secretory cells; this result was unaffected by neuraminidase digestion. DBA was also bound to vascular endothelial cells in vessels of every caliber. Parotid secretory cells did not react with PNA, Neu-PNA, RCA I and LTA. Saponification before enzymatic digestion did not affect the PNA and DBA results. The apical surface of striated duct cells bound all the lectin conjugates with a variable avidity. ConA (Fig. 2) also stained the cytoplasm of striated duct cells; the reaction product was uniform and distibuted in all the cells. UEA I (Fig. 3), LTA, RCA I and PNA marked the cytoplasm of the cells lining striated ducts with a variable intensity ranging from negative to strong staining. The luminal surface of interlobular duct cells reacted with all lectins employed. Cells resembling mucous goblets, localized in the interlobular ducts, reacted intensely with PNA (Fig. 5), WGA. UEA L LTA and less heavily with the other lectins. These reactive cells increased in number in the distal direction making up about 3040% of the epithelial cells lining the larger interlobular ducts. Mandiblliar gland. SBA and WGA (Fig. 6) stained acinar cells with moderate intensity, and WGA positivity was unaffected by neuraminidase digestion. The sequence Neu-PNA (Fig. 8) caused the strongest reaction in the acinar cells. PNA without neuraminidase digestion (Fig. 7) also produced positive staining but of less intensity. Acinar cells were faintly reactive with DBA (Fig. 9) and showed a stronger positivity when the lectin reaction was preceded by enzymatic treatment (Fig. 10). As in the parotid gland, the vascular endothelium reacted with DBA as well. Potassium hydroxide treatment prior to sialidase degradation did not affect the PNA and DBA reactivity. LTA, UEA I and ConA did not mark mandibular acinar cells. Demilunar cells only reacted with Con A, LTA and UEA I. The staining obtained after ConA incubation was homogeneous and discrete (Fig. 11), while LTA and UEA I (Fig. 12) yielded variable positivity in the demilunar cells, ranging from negative to moderate staining.
V. Pedini et al.
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Fig. 1. Parotid gland, SBA. All acinar cells are heavily stained as well as the apical surface of striated re activity is present in secretory duct cells (arrows). x350. Fig. 2. Parotid Parotid gland, ConA. An intense reactivity cells. The cytoplasm of cells linmg striated ducts shows uniform staining. x 350. Fig. 3. Parotid gland, UEA 1. Acinar cells with positive reaction localized in the apical cytoplasm and at the surface. The apex of all striated duct cells shows an intense affinity for this lectin while the cytoplasm exhibits light to heavy staining. x350. Fig. 4. Parotid gland, DBA. Acinar cells stain weakly. Note the endothelium staining of the capillaries (arrows) and a vessel (arrowhead). x 380.
Lectin histochemistry in fallow-deer salivary glands
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Fig. 5. Parotld gland, PNA Apices of cells hning the interlobular duct react with PNA. Some cells also show an intense cytoplasmic positiVity. x 300. Fig. 6. Mandibular gland. WGA. Acinar cells exhibit a moderate staining; demllunes are unreactive (asterisks). The luminal surface of striated duct cells stains intensely (arrow). x350. Fig. 7. Mandibular gland, PNA. A weak staming IS locahzed m the aCInar c ells and apex of cells lining a stnated duct (arrow). Demilunar cells are unstained (asterisks). x 410. Fig. 8. Mandibular gland. Neu-PNA. Siahdase digestion enhance acinar cell reactivity and apIcal positivity of duct cells (arrow) for PNA while demilunes remain unreactive (asterisks). x410
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Fig. Fig. 9. 9. Mandibular gland, DBA. Acinar cells and apices of striated duct cells stain weakly; demilunes are unstained. Vascular endothelial cells react with DBA (arrows). xx410. 410. Fig. 10. Mandibular gland, Neu·DBA. Sialidase digestion promotes increased DBA reactivity in the acinar cells and at the apical Neu-DBA. surface of striated duct cells (arrow). A weak cytoplasmic positivity also appears in these last cells. Demilunes remain unreactive (asterisks). x41O. Fig. 11. Mandibular gland, ConA. Demilunar cells and cells lining striated ducts show an uniform staining throughout the cytoplasm. Acinar cells are unstained. x 350. Fig. 12. Mandibular gland, UEA I. Demilunar cells exhibit a variable staining with UEA I while acinar cells are unreactive. unreactive. Striated duct cells show an intense posItivity at the luminal surface. Some cells present also staining in the supranuclear portion (arrows). (arrows). x350
Lectin histochemistry in fallow-deer salivary glands
87
The apical surface of cells lining all the different segments of the excretory system presented a lectin staining pattern similar to that obtained in the parotid gland. The only difference was the intensification of PNA and DBA reactivity after neuraminidase digestion. In addition to the staining of the luminal surface, striated duct cells showed uniform staining throughout the cytoplasm with the NEU-DBA sequence (Fig. 10), ConA (Fig. 11) and SBA. UEA I caused an intense staining in a few cells lining striated ducts; the reaction product was localized in the supranuclear cytoplasm (Fig. 12). Controls. All staining by the lectins was completely inhibited when the lectin-peroxidase conjugates were incubated with the appropriate specific sugar.
Discussion Parotid and mandibular glands of the fallow-deer are structurally and topographically similar to those of most ruminant species (Saber and Hofmann, 1984). Our previous histochemical investigations of the major salivary glands of fallow-deers (Pedini et aI., 1995) demonstrated that the acinar cells of the mandibular gland mainly possess acid glycoconjugates with sialic acid and carboxyls, while parotid secretory cells and mandibular demilunar cells have neutral glycoproteins in their secretion. These studies also demonstrated the absence of neuraminic acid in parotid secretory cells of the fallow-deer. The lectin histochemistry results reported in the present study confirm these data. The uniform and intense SBA staining in parotid secretory cells of the fallowdeer could be attributed to the homogeneous presence of glycoconjugates containing N-acetylgalactosamine. This sugar residue is particularly present in the p-anomeric form as demonstrated by the weaker result obtained with DBA, a lectin which shows no affinity for p-N-acetylgalactosamine (Schulte et aI., 1985). The faint DBA reactivity and PNA negativity were unaffected by neuraminidase digestion indicating the absence of terminal sialic acid linked to D-galactose-(p1 ~ 3)N-acetylgalactosamine or N-acetylgalactosamine. Parotid glands of others mammals (cat: Accili et aI., 1989; rat. rabbit and hare: Accili et aI., 1992; dog: Pedini et aI., 1994 a) contain these sialoglycoconjugates in their secretion. However. already in 1962. Shackleford and Klapper observed morphological and histochemical differences comparing ruminant parotid glands with the same organ in other mammals. Moreover. lectin histochemical studies have never been carried out on the ruminant parotid gland. The intense reaction obtained with ConA and WGA shows the presence of mannose and N-acetylglucosamine in the acinar cells of the fallow-deer parotid gland. The latter lectin also recognizes sialic acid (Monsigny et aI., 1980) but the WGA staining did not change when sialidase treatment preceded lectin incubation; the absence of neuraminic acid in parotid secretory cells of the fallow-deer has been shown in our previous study (Pedini et aI., 1995). The mucous acinar cells of the mandibular gland contain acidic glycoconjugates with terminal sialic acid residues indicated by their loss of affinity for Alcian blue after neuraminidase digestion (Pedini et al.. 1995). The increased reactivity of PNA and DBA following the enzymatic removal of sialic acid confirms this result, showing the presence of sialic acid linked to D-galactose-(p1 ~ 3)N-acetylgalactosamine and N-acetylgalactosamine, respectively. The mandibular gland of other ruminants (ovine: Schulte et aI., 1985; bovine: Menghi et aI., 1992) contains the terminal trisaccharide sialic acid-D-galactose-CP1 ~ 3)N- acetylgalactosamine and the terminal disaccharide sialic acid-N-acetylgalactosamine in the glycoconjugates secreted by the mucous cells. The failure of saponification before sialidase digestion to increase PNA
88
V. Pedini et aI.
and DBA reactivity indicate the absence or presence of only low levels of sialic acid residues with O-acetyl substituents at C4 (Schulte et aI., 1985). Mandibular acinar cells also showed a moderate affinity for WGA, unaffected by sialidase digestion, which shows the presence of N-acetylglucosamine residues. This sugar is partly present in the dimer D-galactose-(p1 ~ 4)N-acetylglucosamine as indicated by the weak positivity of RCA I. An interesting finding is the demilunar affinity for UEA I, LTA and ConA, which reflects the presence of glycoconjugates with fucose and mannose residues. Serous demilunes lack detectable complex carbohydrate in sheep mandibular gland (Schulte et aI., 1985) and react with DBA and WGA only in the bovine mandibular gland (Menghi et aI., 1992). However, Spicer and Schulte (1992) report in a recent review on cell glycoconjugates that " ... comparative studies have shown marked dissimilarities among genera and species in the glycoconjugates present at a given site. Cells exhibiting such variability have been found in salivary glands, kidney, trachea, and other tissues ..." Cells lining striated and interlobular ducts showed a predominant lectin-affinity at their apical surface, both in the parotid and in the mandibular gland. All the lectins employed produced a positivity of variable intensity at these sites. The only difference between parotid and mandibular gland is the presence of a sialoglycoconjugate in the apical plasmalemma of ductal cells, as demonstrated by the increased PNA and DBA reaction following sialidase digestion in the latter gland. Terminal sialic acid has been found at the apical surface of ductal cells in the mandibular glands of other mammals (Schulte et aI., 1985; Menghi et aI., 1992; Pedini et aI., 1994 b). The cytoplasm of striated duct cells mainly reacted with ConA and UEA I. The positivity of ConA is probably due to the presence of glucose residues in glycogen, which may represent an energy reserve for the production of hypotonic saliva by the striated ducts (Fawcett, 1986; Menghi et al., 1987). Fucosylated glycoconjugates, demonstrated by UEA I affinity, have already been shown in the cytoplasm of some cells lining striated ducts in the ovine mandibular gland (Schulte et aI., 1985). Their presence may be connected to the relative hydrophobicity of this sugar, which could contribute to the transport functions of duct cells. Endothelial cells lining the vasculature had affinity for DBA in both salivary glands examined. DBA binds to the plasma membrane of capillary endothelial cells in a variety of tissues of the mouse (Ponder, 1983), whereas UEA I is considered as a marker for human blood vessels (Damianov, 1987). Further investigations could verify the DBA positivity of endothelial cells in other organs of the fallow-deer.
Acknowledgements The authors wish to thank Mrs Gabriella Mancini for her excellent technical assistance. The authors are also grateful to the president and technical staff of the "Cooperativa Rinascita Montana di Nocera Umbra" for invaluable aid in collecting material. This work was partly supported by grants (40% and 60%) from MURST.
References Accili D, Menghi G, Bondi AM, and Scocco P (1992) Glycoconjugate compOSition of mammalian parotid glands elucidated In situ by lectins and glycosidases. Acta histochem 92: 196-206 Accili D, Menghi G, and Materazzi G (1989) Biochemical analysis and lectin histochemistry for detecting complex carbohydrates in the cat parotid gland. Arch BioI 100: 337-351
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Damianov I (1987) BIOlogy of disease: lectin cytochemistry and histochemistry. Lab Invest 57: 5-18 Fawcett OW (1986) A textbook of histology. Saunders WB. Philadelphia. pp 593-597 Menghi G. Accili D. and Bondi AM (1987) Differential binding sites of peroxidase-labelled lectins In the submandibular gland of sucking and adult cats. Acta histochem 82: 63-75 Menghl G. Accili D. Scocco P. and Materazzi G (1992) Sialoglycoderivatives of bOVine submandibular gland Identified in situ by histochemical techmques combined with lectins. HistochemIstry 97: 397-403 Menghi G. and Materazzl G (1994) Exoglycosidases and lectlns as sequencing approaches of salivary gland olIgosaccharides. Histol Histopath 9: 173-183 Monsigny M. Roche AC, Sene C, Maget-Dana R. and Delmotte F (1980) Sugars-lectin interactions: how does wheat germ agglutinin bind sialoglycoconJugates. Eur J Biochem 104: 147-152 Moschera1. and Pigman W (1975) The isolation and characterization of rat sublingual mucus glycoprotein. Carbohydr Res 40' 53-67 Nleuw Amerongen AV. Bolscher JGM. and Veerman ECI (1995) Salivary mucins. protective functIons In relation to their diverSity. Glycobiol 5: 773- 740 Pedlm V. Ceccarelli P. and GargIUlo AM (1994 a) LocalizatIon of glycoconJugates in dog parotid gland by lectin histochemistry. Vet Res Comm 18: 269-279 Pedini V. CeccarellI P. and Gargiulu AM (1994 b) Glycoconjugates in the mandibular salIvary gland of adult dogs revealed by lectin histochemistry. Res Vet SCI 57: 353-357 Pedlni V. Fagioli 0, and Lorvik S (1995) Histochemical stud on the major salivary glands of fallow deer (Dama dama L.). Atti SISVet. in press Ponder BAJ (1983) Lectin histochemistry. In: Polak 1M. Van Noorden S (Eds) Immunocytochemistry: practIcal applicatIOns In pathology and biology. Wright PSG. Bristol. pp 129- 142 Saber AS. and Hofmann RR (1984) Comparallve anatomical and topographic studies of the salivary glands of red deer (Cervus elaphus). fallow deer (Cervus dama) . and mouflon (OVIS ammon ml/simon) - Ruminantia. Cervidae. Bovidae. Gegenbaurs morph lahrb 130: 273-286 Schulte BA. Spicer SS. and Miller RL (1985) Lectin histochemistry of secretory and cell-surface glycoconjugates in the ovine submandibular gland. Cell Tissue Res 240: 57-66 Shackleford JM. and Klapper CE (1962) Structure and carbohydrate histochemistry of mammalian salivary glands. Am 1 Anat 111: 25-47 Spicer SS. and Schulte BA (1992) Diversity of cell glycoconJugates shown histochemically: a perspectlve. l Hlstochem Cytochem 40: 1- 38 Tabak LA (1995) In defense of the oral cavity: structure. biosynthesis. and function of salivary muclns. Ann Rev Physiol 57: 547- 564