FULL PAPER Anatomy
Lectin Histochemical Studies on the Vomeronasal Organ of the Sheep Dalia IBRAHIM1,2), Nobuaki NAKAMUTA1,3), Kazumi TANIGUCHI4) and Kazuyuki TANIGUCHI1,3)* 1)United
Graduate School of Veterinary Sciences, Gifu University, 1–1 Yanagido, Gifu, Gifu 501–1193, Japan of Histology, Faculty of Veterinary Medicine, South Valley University, Qena 83523, Egypt 3)Laboratory of Veterinary Anatomy, Faculty of Agriculture, Iwate University, 3–18–8 Ueda, Morioka, Iwate 020–8550, Japan 4)Laboratory of Veterinary Anatomy, School of Veterinary Medicine, Kitasato University, Towada, Aomori 034-8628, Japan 2)Department
(Received 6 December 2012/Accepted 2 April 2013/Published online in J-STAGE 16 April 2013) ABSTRACT.
The vomeronasal organ of sheep was examined using lectin histochemistry in order to compare the types and amounts of the glycoconjugates among various components of the vomeronasal sensory and non-sensory epithelia. In the vomeronasal sensory epithelium, Dolichos biflorus agglutinin (DBA) stained particular cells, located at the same level as the vomeronasal receptor cells, while the distribution, shape and number of the stained cells did not correspond to those of the vomeronasal receptor cells. Datura stramonium lectin (DSL), Concanavalin A (Con A), Phaseolus vulgaris agglutinin-E (PHA-E) and Phaseolus vulgaris agglutinin-L (PHA-L) labeled the basal cells of both vomeronasal sensory and non-sensory epithelia. While, Wheat germ agglutinin (WGA), Succinylated-wheat germ agglutinin (sWGA), Lycopersicon esculentum lectin (LEL), Solanum tuberosum lectin (STL) and Ricinus communis agglutinin-I (RCA-120) labeled the basal cells of the sensory epithelium, and Bandeiraea simplicifolia lectin-I (BSL-I) stained the basal cells of the non-sensory epithelium, respectively. Seventeen lectins labeled the free border of both vomeronasal sensory and non-sensory epithelia, while Sophora japonica agglutinin (SJA), Jacalin and Pisum sativum agglutinin (PSA) labeled neither free border of the sensory nor that of non-sensory epithelia. The expression pattern of glycoconjugate was similar, but not identical, in the free border between the sensory and non-sensory epithelia. These results indicate that there are dissimilar features in the type and amount of glycoconjugates between the vomeronasal sensory and non-sensory epithelia, and at the same time, among the various cell types either in the vomeronasal sensory or non-sensory epithelium. KEY WORDS: glycoconjugates, olfactory system, ruminants. doi: 10.1292/jvms.12-0532; J. Vet. Med. Sci. 75(9): 1131–1137, 2013
The majority of mammalian species as well as many reptiles and amphibians possess the second olfactory system, the accessory olfactory or vomeronasal system, which detects particular classes of chemical signals, such as pheromones [6, 13, 15, 16, 29, 30]. The vomeronasal organ (VNO) is the receptor organ of the accessory olfactory system, a neural pathway exposed to the external environment, and sends information by axons directly to the brain areas related with reproductive and maternal behaviors [2, 3]. The VNO has been studied in many species including horses, sheep, goats, cows, dogs, cats, bats, mink, mice and human [2, 3, 5, 8, 14]. The VNO complex includes the epithelial tubes and several related structures, such as glands, nerves and blood vessels [11, 26]. In sheep, a pair of VNOs is located on both sides of the base of the nasal septum. A lamina of cartilage enwraps the VNO and its lamina propria including the vomeronasal glands, vessels and nerve bundles of each side. The anterior part of the vomeronasal cartilage is connected to the cartilage of the incisive duct. Vomeronasal receptors are distributed along the sensory epithelium, which occupies the medial wall of the VNO [18, 25, 27]. The histological and histochemical *Correspondence to: Taniguchi, K., Laboratory of Veterinary Anatomy, Faculty of Agriculture, Iwate University, 3–18–8 Ueda, Morioka, Iwate 020–8550, Japan. e-mail:
[email protected] ©2013 The Japanese Society of Veterinary Science
features of the sheep VNO have been evaluated before by some authors: Kratzing [18] focused on the ultrastructure of the sheep vomeronasal sensory epithelium, while Salazar et al. [27, 28] studied the lectin binding patterns in the VNO with special reference to nerve bundles and blood vessels, respectively. However, reevaluation of the sheep VNO by histochemical and lectin histochemical techniques enabled us to compare the glycoconjugates content in the vomeronasal sensory and non-sensory epithelia and to build the base for our future study by discussing the differences there-in between various sheep breeds. Lectins are proteins that bind to glycoconjugates non-immunologically. They are considered to be the major analytic tool for the study of both soluble and cellular glycoconjugates. They are mainly specific to the terminal carbohydrates of sugar chains [7] and are extensively used for the differentiation of cells according to their glycoconjugate contents on the histological sections [9, 17, 19, 21, 22, 24]. In this study, the VNO of the adult Corriedale sheep was examined by lectin histochemistry to reveal the binding patterns with the glycoconjugates in both the vomeronasal sensory and non-sensory epithelia of the sheep. MATERIALS AND METHODS Animals: Three adult, castrated Corriedale sheep were studied. They were obtained from Kitasato University (Aomori, Japan). Animals were euthanized by the perfusion of physiological saline through the common carotid arter-
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D. IBRAHIM, N. NAKAMUTA, K. TANIGUCHI AND K. TANIGUCHI Table 1. Binding specifities of lectins used in this study Lectins Wheat germ agglutinin Succinylated-wheat germ agglutinin Lycopersicon esculentum lectin Solanum tuberosum lectin Datura stramonium lectin Bandeiraea simplicifolia lectin-II Dolichos biflorus agglutinin Soybean agglutinin Bandeiraea simplicifolia lectin-I Vicia villosa agglutinin Sophora japonica agglutinin Ricinus communis agglutinin-I Jacalin Peanut agglutinin Erythrina cristagalli lectin Ulex europaeus agglutinin-I Concanavalin A Pisum sativum agglutinin Lens culinaris agglutinin Phaseolus vulgaris agglutinin-E Phaseolus vulgaris agglutinin-L
Abbreviation WGA s-WGA LEL STL DSL BSL-II DBA SBA BSL-I VVA SJA RCA-120 PNA ECL UEA-I Con A PSA LCA PHA-E PHA-L
Concentration (mg/ml) 10ˉ2
1.0 × 1.0 × 10ˉ2 2.0 × 10ˉ2 1.0 × 10ˉ2 4.0 × 10ˉ3 4.0 × 10ˉ3 1.0 × 10ˉ2 1.0 × 10ˉ2 4.0 × 10ˉ3 4.0 × 10ˉ3 2.0 × 10ˉ2 2.0 × 10ˉ3 5.0 × 10ˉ4 4.0 × 10ˉ3 2.0 × 10ˉ2 2.0 × 10ˉ2 3.3 × 10ˉ3 4.0 × 10ˉ3 4.0 × 10ˉ3 5.0 × 10ˉ3 2.5 × 10ˉ3
Binding specificity GlcNAc, NeuAc (GlcNAc)n (GlcNAc)2–4 (GlcNAc)2–4 (GlcNAc)2–4 GlcNAc Gal, GalNAc Gal, GalNAc Gal, GalNAc Gal, GalNAc Gal, GalNAc Gal, GalNAc Gal, GalNAc Gal Gal, GalNAc Fuc Man, Glc Man, Glc Man, Glc Oligosaccharide Oligosaccharide
Fuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; GlcNAc, N-acetylglucosamine; Man, mannose; NeuAc, N-acetylneuraminic acid.
ies under deep anesthesia with intravenous injection of the sodium pentobarbital at a dose of 20 mg/kg body weight. All procedures were in accordance with the Principles for Animal Experiments of Iwate University and approved by the intramural committee for the care and use of animals. Animals were perfused with Bouin’s solution without acetic acid via common carotid arteries. After decapitation, the nasal parts were immersed in the same fixative for 2 days and then decalcified in 10% ethylenediaminetetraacetic acid for several months, routinely embedded in paraffin and cut transversally at 5 µm. Some sections were stained with hematoxylin-eosin, periodic acid/Schiff (PAS) or alcian blue (pH 2.5) for histological examinations. Lectin histochemistry: The other sections were processed for histochemistry with the avidin-biotin complex (ABC) method using 21 types of biotinylated lectins (Table 1) in the lectin screening kits I-III (Vector Laboratories, Burlingame, CA, U.S.A.). After deparaffinization and rehydration, sections were incubated with 0.3% H2O2 in methanol to eliminate endogeneous peroxidase activity. Sections were rinsed in 0.01 M phosphate buffered saline (PBS, pH 7.4) and incubated with 1% bovine serum albumin to block nonspecific reactions. After rinsing, sections were incubated with biotinylated lectins at 4°C overnight and reacted with ABC reagent (Vector Laboratories) at room temperature for 30 min. Thereafter, sections were colorized with 0.05 M Tris-HCl (pH 7.4) containing 0.006% H2O2 and 0.02% 3-3´-diaminobenzidine tetrahydrochloride. Control stainings were made by using of PBS to replace lectins.
RESULTS Topographical and histological features of the VNO: The VNO was a pair of tubes lined by epithelium situated at the base of the nasal septum (Fig. 1A). The VNO and its associated lamina propria were surrounded by an incomplete ring of vomeronasal cartilage which was hyaline in nature. The nasal mucosa covered the VNO laterally. The lamina propria of the VNO included associated glands, connective tissue, nerves and vessels which were organized to surround the VNO. The vomeronasal glands as well as the vomeronasal cartilage reacted intensely positive to PAS and alcian blue. The excretory ducts of the glands opened into both the lateral and medial sides of the vomeronasal epithelium. Three to 7 large veins with relatively thick walls were situated lateral to the VNO. The sensory epithelium covered the ventral twothirds of the medial side of the VNO, while the non-sensory epithelium covered the lateral side and the dorsal one-third of the medial side of the VNO. Some melanin pigments were found both in the basal region of the vomeronasal sensory epithelium and the underlying lamina propria, but they were not equally distributed along the vomeronasal sensory epithelium (Fig. 1B and 1C). The vomeronasal sensory epithelium of the Corriedale sheep was composed of three types of cells: supporting, receptor and basal. Each type of cell had properties that have been already described for the cells of vomeronasal sensory epithelium in sheep [18, 27, 28] and other ruminant species: Angora goats [4], buffaloes [1] and cows [26]. Briefly, nuclei of the vomeronasal basal cells, receptor cells and supporting
LECTIN BINDING IN SHEEP VOMERONASAL ORGAN
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Fig. 1. Topographical and histological features of the vomeronasal organ in the Corriedale sheep. (A) Transverse section of the vomeronasal organ stained with periodic acid Schiff (PAS). The left side of the figure is medial, and the upper, dorsal. Arrowhead indicates the melanin pigments. Bar=400 µm. Higher magnification of the vomeronasal sensory epithelium stained with PAS (B) and Alcian blue (C). Arrowheads indicate PAS-positive, non-alcianophilic supranuclear granules in the cytoplasm of the vomeronasal receptor cells. Bars=30 µm. Higher magnification of the vomeronasal non-sensory epithelium stained with PAS (D) and Alcian blue (E). Arrowheads indicate goblet cells. Bar=50 µm. V, large veins; VNC, vomeronasal cartilage; VNG, vomeronasal gland; VNO, lumen of the vomeronasal organ.
Fig. 2. Vomeronasal sensory epithelium reacted with four lectins: LEL (A), PHA-L (B), VVA (C) and DBA (D). Sections are counterstained with methyl green in (B) and (C). Arrowheads in (A) indicate vomeronasal receptor cells. Arrowheads in (B) indicate supporting cells, and the arrow indicates basal cells. Arrowheads in (C) indicate supranuclear cytoplasmic granules in the receptor cells. Arrowheads in (D) indicate unrecognized spindle-shaped cells labeled by DBA, and the arrow indicates a region in the free border stained with DBA. Bar=30 µm in (A)-(D).
cells were situated in the basal, middle and apical regions of the vomeronasal epithelium, respectively. The vomeronasal basal cells had scanty cytoplasm and oval or round nuclei,
which were pale with a distinct nucleolus and were smaller in size than those of the receptor and supporting cells. The vomeronasal receptor cells were pear-shaped and extended their dendrites to the luminal surface and their axons toward the basal lamina. The nuclei of receptor cells were arranged in one layer. The cytoplasm was accompanied by PASpositive non-alcianophilic supranuclear granules (Fig. 1B and 1C). The vomeronasal supporting cells had dark, oval nuclei arranged in two to four layers in the upper region of the vomeronasal sensory epithelium. The non-sensory epithelium was lined by pseudostratified ciliated columnar epithelium with goblet cells. The goblet cells reacted intensely positive with both PAS and alcian blue (Fig. 1D and 1E). Lectin binding patterns in the vomeronasal sensory epithelium: the receptor cells of the VNO were stained with varying intensity among 5 of the 21 lectins used in this study: Wheat germ agglutinin (WGA), Succinylated-wheat germ agglutinin (s-WGA), Lycopersicon esculentum lectin (LEL), Solanum tuberosum lectin (STL) and Concanavalin A (Con A) (Table 2). Cell processes, cell membranes and perinuclear cytoplasm were stained intensely, but the nuclei were not stained (Fig. 2A). Six lectins: DSL, Bandeiraea simplicifolia lectin-II (BSL-II), Bandeiraea simplicifolia lectin-I (BSL-I), Ricinus communis agglutinin-I (RCA-120), Peanut agglutinin (PNA) and Ulex europaeus agglutinin-I (UEA-I), faintly stained the perinuclear cytoplasm of the receptor cells. Eight lectins: Soybean agglutinin (SBA), Sophora japonica agglutinin (SJA), Jacalin, Erythrina cristagalli lectin (ECL), Pisum sativum agglutinin (PSA), Lens culinaris agglutinin (LCA), Phaseolus vulgaris agglutinin-E (PHA-E) and Phaseolus vulgaris agglutinin-L (PHA-L), did not label the vomeronasal receptor cells (Fig. 2B). Vi-
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Table 2. Lectin binding patterns of the sensory epithelium in the vomeronasal organ of sheep Lectin
Free Border
Receptor cells
Supporting cells
Basal cells
WGA s-WGA LEL STL DSL BSL-II DBA SBA BSL-I VVA SJA RCA-120 Jacalin PNA ECL UEA-I Con A PSA LCA PHA-E PHA-L
++ + ++ ++ ++ + ++or−* ++ + ++ − ++ − ++ ++ ++ ++ − − ++ ++
+ + + + +/− +/− − − +/− #+ − +/− − +/− − +/− + − − − −
+/− +/− +/− +/− + +/− − − − +/− − + − − − +/− + − − + +
+/− +/− +/− +/− ++ − − − − − − + − − − − + − − ++ ++
−, negative staining; +/−faint staining; +, moderate staining; ++, intense staining; *, the staining reaction is not equally distributed along the wall of the vomeronasal sensory epithelium; #+, supranuclear granules in the cytoplasm of the receptor cells.
cia villosa agglutinin (VVA) labeled cytoplasmic granules in the supranuclear region of the receptor cells (Fig. 2C). DBA stained a number of spindle-shaped cells (1-7 cells per transverse section of the VNO). These cells were located at the same level as the receptor cells, although the distribution, shape and number of the stained cells did not correspond to those of the receptor cells (Fig. 2D). Twelve out of the 21 lectins used, but not DBA, SBA, BSL-I, SJA, Jacalin, PNA, ECL, PSA nor LCA, labeled the vomeronasal supporting cells with varying intensity (Table 2). The staining patterns were similar to each other and were limited to the cytoplasm of the supporting cells (Fig. 2B). Seven lectins: WGA, s-WGA, LEL, STL, BSL-II, VVA and UEA-I, faintly stained the cytoplasm of the supporting cells. Nine lectins: WGA, s-WGA, LEL, STL, DSL, RCA-120, Con A, PHA-E and PHA-L, stained the basal cells of the vomeronasal sensory epithelium with varying intensity (Table 2). The staining patterns were similar to each other and were limited to the cytoplasm and cell membrane of the basal cells (Fig. 2B). No lectins labeled the nuclei of the basal cells. Other than SJA, Jacalin, PSA and LCA, all the lectins used in this study stained the free border of the vomeronasal sensory epithelium with varying intensity (Table 2). DBA labeled small regions in the free border of the vomeronasal sensory epithelium (Fig. 2D) that were not related to the distribution, number or shape of the receptor cells or even
the supporting cells of the vomeronasal sensory epithelium. Lectin binding patterns in the vomeronasal non-sensory epithelium: Out of the 21 lectins used in this study, ten lectins: WGA, s-WGA, LEL, STL, BSL-II, BSL-I, VVA, RCA-120, ECL and UEA-I, stained goblet cells in the vomeronasal non-sensory epithelium with varying intensity (Table 3). The reaction products were localized to the apical cytoplasm containing mucin granules (Fig. 3A). For BSL-I, the staining intensity was different among individuals. It was expressed negatively in some samples and intensely positive in the others. Both DBA and SBA stained the goblet cells intensively in a small, invaginated area of the vomeronasal non-sensory epithelium, while they did not label the remaining goblet cells (Fig. 3B). Nine lectins, DSL, SJA, Jacalin, PNA, Con A, PSA, LCA, PHA-E and PHA-L, did not label the goblet cells at all (Fig. 3C). Among the 21 lectins used in this study, five lectins: DSL, BSL-I, Con A, PHA-E and PHA-L, stained the basal cells in the vomeronasal non-sensory epithelium with varying intensity (Table 3). They stained the cytoplasm of the basal cells, but not the nuclei (Fig. 3D). DBA and SBA stained the basal cells in an invaginated region of the vomeronasal nonsensory epithelium, while they did not stain the remaining basal cells. Two lectins, UEA-I and Con A, faintly stained the apical cytoplasm of the ciliated cells of the vomeronasal nonsensory epithelium (Table 3). Fifteen lectins: WGA, s-WGA, LEL, STL, DSL, BSL-I, VVA, RCA-120, PNA, ECL, UEA-I, Con A, LCA, PHAE and PHA-L, labeled the free border of the vomeronasal non-sensory epithelium with varying intensity (Fig. 3E and 3F). For DBA and SBA, staining was limited to invaginated regions in the free border of the vomeronasal non-sensory epithelium (Fig. 3B). The vomeronasal glands: Histologically, the vomeronasal glands were tubuloacinar units that reacted strongly positive to PAS staining (Fig. 4A). The vomeronasal gland acini had a relatively narrow lumen and consisted of 5 to 7 cuboidal or columnar cells. The nuclei were slightly round to oval in shape and situated basally. The apical cytoplasm was filled with mucin granules. Five out of the 21 lectins used: s-WGA, BSL-II, SJA, Jacalin and LCA, did not stain the vomeronasal gland, while the remaining 16 lectins stained the vomeronasal gland with varying intensity (Table 3). DBA and SBA stained the glandular acinar cells and duct cells intensely (Fig. 4B). VVA staining was intense with the acinar cells. On the other hand, only some of the duct cells were stained by VVA, while the other cells remained unstained (Fig. 4C). PNA labeled only the luminal surface of a few number of the duct cells and faintly stained the acinar cells (Fig. 4D). DISCUSSION We detected the PAS-positive non-alcianophilic supranuclear cytoplasmic granules in the receptor cells of the vomeronasal sensory epithelium. As far as we know, this is the first report to demonstrate these PAS-positive granules.
LECTIN BINDING IN SHEEP VOMERONASAL ORGAN Table 3. Lectin binding patterns of the non-sensory epithelium in the vomeronasal organ of sheep Lectin
Free Border
Goblet cells
Basal cells
WGA s-WGA LEL STL DSL BSL-II DBA SBA BSL-I VVA SJA RCA-120 Jacalin PNA ECL UEA-I Con A PSA LCA PHA-E PHA-L
++ + ++ + ++ − ++or−* ++or−* −~++ ++ +/− + +/− + + + + +/− + ++ ++
++ ++ ++ + − ++ ++or* ++or−* + + − + − − + + − − − − −
− − +/− − ++ − ++or−* ++or−* ++ ++or−* − +/− − − − − + − − ++ ++
Ciliated Vomeronacells sal gland − − − − − − − − − − − − − − − +/− +/− − − − −
+ − + + + +/− ++ ++ + ++ − + − +/− +/− + + +/− − +/− +
−, negative staining; +/−, faint staining; +, moderate staining; ++, intense staining; *, the staining reaction is not equally distributed along the wall of the vomeronasal non-sensory epithelium; −~++, the staining intensity was different among individuals.
The ultrastructural study on sheep made by Kratzing [18] did not mention the presence of such granules, while he found well-developed Golgi apparatus close to the nucleus of the vomeronasal receptor cells and an extensive array of rough endoplasmic reticulum filling most of the perinuclear cytoplasm. The presence of PAS-positive granules in the supranulear region of the receptor cells suggests that the glycoconjugates are vigorously synthesized in the receptor cells than in other types of cells or that the glycoconjugates that show strong affinity for PAS temporarily during the processing in the Golgi apparatus/rough endoplasmic reticulum are synthesized in the receptor cells. On the other hand, some melanin pigments were found both in the basal region of the vomeronasal sensory epithelium and the underlying lamina propria, but they were not equally distributed along the vomeronasal sensory epithelium. The precise location of these melanin pigments has yet to be established. The significance of these pigments is unknown at present; and as far as we know, such melanin pigments have never been reported in sheep [18, 27, 28] nor other ruminants: Angora goats [4], buffaloes [1] and cows [26]. In the vomeronasal sensory epithelium, DBA stained a few number of spindle-shaped cells. Nuclei of the DBA-positive cells were located in the same level as those of the receptor cells. However, the arrangement, shape and frequency of the stained cells did not correspond with those of the receptor
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cells. At the same time, DBA labeled small regions in the free border of the vomeronasal sensory epithelium. The histological appearance of the DBA-positive cells reminds us of a type of cell found in the mouse and rat VNO [10, 12, 20, 23]. According to these previous reports, these cells were described as solitary chemosensory cells (SCCs). SCCs in the mouse and rat have been detected using neural markers, such as the transient receptor potential channel type M5 (TRPM5) or phospholipase C (PLC) β2 [10, 20]. Although we have not done double-labeling experiments, the DBAlabeled cells seemed to react immunohistochemically with anti-PLCβ2 antibody in our preliminary experiment. In the vomeronasal non-sensory epithelium, both DBA and SBA intensely stained the goblet cells in an invaginated area, while they did not stain the other goblet cells. Kratzing [18] found a crypt-like formation lined by ciliated cells and mucous-producing cells in the wall of vomeronasal sensory epithelium, although there were no glands in the lamina propria underlying this area. In contrast, we detected an invaginated area in the non-sensory epithelium with numerous vomeronasal glands in the underlying lamina propria. Neither Kratzing nor our team could find any direct connection between the invaginated areas and the end pieces of the glands, but the staining pattern and intensity of all lectins used in this study were the same in both these invaginated areas and the vomeronasal gland end pieces, suggesting that these invaginated areas are the openings of the vomeronasal glands. DSL, Con A, PHA-E and PHA-L labeled the basal cells of both vomeronasal sensory and non-sensory epithelia. While, WGA, s-WGA, LEL, STL and RCA-120 labeled the basal cells of the vomeronasal sensory epithelium and BSL-I labeled the basal cells of the non-sensory one. The expression pattern of the glycoconjugates was similar and was limited to the cell membrane and cytoplasm of the basal cells. The free border of both vomeronasal sensory and non-sensory epithelia was labeled by seventeen lectins, except for SJA, Jacalin, PSA and LCA in the sensory and SJA, Jacalin, PSA and BSL-II in the non-sensory epithelium. The expression pattern of glycoconjugate was similar, but not identical, in the free border between the sensory and non-sensory epithelia. The lectin staining in the free border may come from the glycoconjugates in the mucus covering the surface of the epithelium and that expressed in the cell membrane of the cilia or the microvilli of the goblet, ciliated, receptor or supporting cells. This study revealed that the goblet cells possibly secrete mucus containing GlcNAc ascendant glycoconjugates, because of the intense reactivity for WGA, s-WGA and LEL. On the other hand, the vomeronasal gland secretes mucus containing Gal or GalNAc ascendant glycoconjugates, because of the intense reactivity for DBA, SBA and VVA. For SBA and VVA, both lectins showed intense staining in the free border of the sensory epithelium. However, for SBA, its reactivity for the receptor cells and the supporting cells is negative. That means there is a possibility that intense SBA reactivity in the free border of the sensory epithelium comes from the secretion from the vomeronasal gland. Interestingly, SBA reactivity in the free
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Fig. 3. Vomeronasal non-sensory epithelium reacted with BSL-II (A), DBA (B), DSL (C), BSL-I (D), LEL (E) and VVA (F). Sections are counterstained with methyl green in (B), (C) and (F). Arrowheads in (A), (C), (E) and (F) indicate goblet cells in a non-invaginated part of the epithelium. Arrowheads in (B) indicate goblet cells of an invaginated part of the non-sensory epithelium. Arrow in (B)-(D) indicates basal cells. Bar=50 µm in (A) and 30 µm in (B)-(F).
border of the non-sensory epithelium is restricted, although VVA, that stains the vomeronasal gland intensely, stains the free border of the non-sensory epithelium intensely. These results suggest the existence of the unsolved mechanism regulating the distribution of the secretory product from the vomeronasal gland. That is, the mechanism that prohibits the certain secretory product from the vomeronasal gland (Gal or GalNAc ascendant glycoconjugates recognized by SBA, not VVA in this study) from staying in the free border of the non-sensory epithelium and thus accelerates the movement and the distribution of it in the free border of the sensory epithelium, resulting in affecting the pheromone perception in the sensory epithelium. For other secretory product from the vomeronasal gland (like Gal or GalNAc ascendant glycoconjugates recognized by DBA), it may also be prohibited from residing in the free border of the non-sensory epithelium and may remain in the cavity and play some functions in the pheromone perception. Such a mechanism is possible to be regulated by the goblet cells, which shows a different nature of glycoconjugates from that in the vomeronasal gland (GlcNAc vs Gal;GalNAc). DBA and SBA, which recognize Gal and GalNAc series of glycoconjugates, also bind to the
Fig. 4. Vomeronasal glands stained with PAS stain (A) or lectins: DBA (B), VVA (C) and PNA (D). The arrangement of the vomeronasal glands and vomeronasal ducts around the vomeronasal organ is shown. In all figures, arrowheads indicate acini of the vomeronasal glands, and arrows indicate ducts of the vomeronasal glands. NB, nerve bundle; V, vein. Bar=50 µm in (A)-(D).
goblet cells, but the reacted goblet cells are small number and in the restricted area. There is a possibility that these goblet cells regulate the resident rate of certain Gal;GalNAc ascendant glycoconjugates from the vomeronasal gland in the non-sensory epithelium. In addition, BSL-II stains the goblet cells intensely, but its reactivity in the free border of the non-sensory epithelium is negative, and that of the sensory epithelium is moderate; therefore, there is a possibility that the goblet cells may also concern the function of the vomeronasal organ via another unsolved mechanism, beyond simply secreting the covering mucus. This study revealed the significant difference of the glycoconjugates between the goblet cells and the vomeronasal gland, suggesting the idea that the goblet cells in the non-sensory epithelium also contribute the function of the vomeronasal organ, the perception of the pheromone in the sensory epithelium, via an unsolved mechanism, and a further study including the nature of the goblet cells also should be conducted for further understanding the function of the vomeronasal system. ACKNOWLEDGMENT. This work was supported by Grant-in-Aid for Graduate Students from The United Graduate School of Veterinary Science, Gifu University (D.I.). REFERENCES 1. Abbasi, M. 2007. The vomeronasal organ in buffalo. Ital. J. Anim. Sci. 6: 991–994. 2. Adams, D. R. and Wiekamp, M. D. 1984. The canine vomerona-
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