animals were euthanized with 
 intravenous injection of sodium pentobarbital at a dose of 20 mg / kg body weight and then 
 perfused with physiological saline through the common carotid arteries under deep 
 anesthesia . 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 the bouin s solution without acetic acid via the 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 m . some sections were 
 stained with hematoxylin - eosin ( h&e ) , periodic acid / schiff ( pas ) , alcian blue ( ph 1.0 
 and 2.5 ) or crossmon s trichrome 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 1table 1.binding specifities of lectins used in this studylectinsabbreviationconcentration ( mg / ml)binding specificitywheat germ agglutininwga1.0  10glcnac , neuacsuccinylated - wheat germ agglutinins - wga1.0  10(glcnac ) nlycopersicon esculentum lectinlel2.0  10(glcnac ) 24solanum tuberosum lectinstl1.0  10(glcnac ) 24datura stramonium lectindsl4.0  10(glcnac ) 24bandeiraea simplicifolia 
 lectin - iibsl - ii4.0  10glcnacdolichos biflorus agglutinindba1.0  10gal , galnacsoybean agglutininsba1.0  10gal , galnacbandeiraea simplicifolia 
 lectin - ibsl - i4.0  10gal , galnacvicia villosa agglutininvva4.0  10gal , galnacsophora japonica agglutininsja2.0  10gal , galnacricinus communis agglutinin - irca-1202.0  10gal , galnacjacalin5.0  10gal , galnacpeanut agglutininpna4.0  10galerythrina cristagalli lectinecl2.0  10gal , galnaculex europaeus agglutinin - iuea - i2.0  10fucconcanavalin acona3.3  10man , glcpisum sativum agglutininpsa4.0  10man , glclens culinaris agglutininlca4.0  10man , glcphaseolus vulgaris agglutinin - epha - e5.0  10oligosaccharidephaseolus vulgaris agglutinin - lpha - l2.5  10oligosaccharidefuc , fucose ; gal , galactose ; galnac , n - acetylgalactosamine ; glc , glucose ; glcnac , 
 n - acetylglucosamine ; man , mannose ; neuac , n - acetylneuraminic acid . ) in the lectin screening kits i - iii ( vector laboratories , burlingame , ca , 
 u.s.a . ) . after deparaffinization and rehydration , the sections were incubated with 0.3% 
 h2o2 in methanol to eliminate endogenous peroxidase activity . the 
 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 , the sections were 
 incubated with biotinylated lectins at 4c overnight and reacted with abc reagent ( vector 
 laboratories ) at room temperature for 45 min . thereafter , the sections were colorized with 
 0.05 m tris - hcl ( ph 7.4 ) containing 0.006% h2o2 and 0.02% 
 33-diaminobenzidine tetrahydrochloride , dehydrated , cleared and mounted . fuc , fucose ; gal , galactose ; galnac , n - acetylgalactosamine ; glc , glucose ; glcnac , 
 n - acetylglucosamine ; man , mannose ; neuac , n - acetylneuraminic acid . topographical and histological features of the olfactory mucosa : the 
 olfactory mucosa occupied only a small region at the caudal roof of the nasal cavity , where 
 it entirely covered the ethmoturbinates and the caudal parts of the dorsal and middle nasal 
 conchae . the olfactory mucosa consisted of the olfactory epithelium and the underlying 
 lamina propria ( fig . ( a ) transverse section of the 
 olfactory mucosa stained with periodic acid schiff ( pas ) . bar=100 m . 
 higher magnification of the olfactory epithelium stained with ( b ) pas and ( c ) alcian 
 blue ( ph 2.5 ) . higher magnification of the bowman s glandular acini stained with 
 hematoxylin and eosin ( d ) , alcian blue ( ph 2.5 ) ( e ) or crossmon s trichrome ( f ) . arrowheads indicate bowman s glandular acinar cells . a , artery ; bg , bowman s glands ; 
 nb , nerve bundle ; obc , olfactory basal cells ; orc , olfactory receptor cells ; osc , 
 olfactory supporting cells . each 
 type of cell had characteristics that have been already described for the cells of the 
 olfactory epithelium in sheep [ 9 , 13 , 20 , 22 ] . briefly , nuclei of the olfactory basal , receptor and 
 supporting cells were situated in the basal , middle and apical regions of the olfactory 
 epithelium , respectively ( fig . their nuclei were pale 
 with distinct nucleolus and were smaller than those of the receptor and supporting cells . the olfactory 
 receptor cells were pear - shaped and extended their dendrites to the luminal surface and 
 their axons toward the basal lamina . the 
 olfactory supporting cells had dark , oval - shaped nuclei arranged in three or four of the 
 layers in the upper region of the olfactory epithelium . some melanin pigments were found 
 both in the basal region of the olfactory epithelium and the underlying lamina propria , but 
 they were not equally distributed along the olfactory epithelium ( fig . ( a ) transverse section of the 
 olfactory mucosa stained with periodic acid schiff ( pas ) . bar=100 higher magnification of the olfactory epithelium stained with ( b ) pas and ( c ) alcian 
 blue ( ph 2.5 ) . higher magnification of the bowman s glandular acini stained with 
 hematoxylin and eosin ( d ) , alcian blue ( ph 2.5 ) ( e ) or crossmon s trichrome ( f ) . arrowheads indicate bowman s glandular acinar cells . a , artery ; bg , bowman s glands ; 
 nb , nerve bundle ; obc , olfactory basal cells ; orc , olfactory receptor cells ; osc , 
 olfactory supporting cells . the secretory end - pieces of the bowman s glands were of the tubuloacinous type , and 
 their ducts extended vertically through the olfactory epithelium to open into the free 
 border of the olfactory epithelium . acini of the bowman s glands consisted of pyramidal 
 cells containing basally situated oval or round nuclei and surrounding a narrow lumen . brownish granules were detected in the basal cytoplasm of the acinar cells as well as their 
 duct cells ( fig . 1e ) stained the apical cytoplasm of the acinar cells , while 
 crossmon s trichrome ( fig . lectin binding patterns in the olfactory mucosa : the receptor cells of the 
 olfactory epithelium were labeled , with varying intensity , among 12 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 ) , datura stramonium lectin ( dsl ) , soybean agglutinin ( sba ) , 
 bandeiraea simplicifolia lectin - i ( bsl - i ) , vicia villosa 
 agglutinin ( vva ) , ricinus communis agglutinin - i ( rca-120 ) , concanavalin a 
 ( con a ) , pisum sativum agglutinin ( psa ) and phaseolus 
 vulgaris agglutinin - e ( pha - e ) . cell processes , cell membranes and perinuclear 
 cytoplasm were intensely stained , but the nuclei were not stained ( fig . 2a , 2afig . 2.olfactory mucosa reacted with six lectins : lel ( a ) , ( a ) , stl ( b ) , ( b ) , vva ( c ) , 
 ( c ) , lca ( d ) , ( d ) , pna ( e ) , ( e ) and pha - l ( f ) and ( f ) . bar=50 m in ( a)(f ) and 30 m 
 in ( a)(f). , 2b and 2b ) . three lectins , s - wga , sba and psa , faintly stained the perinuclear 
 cytoplasm of the receptor cells . punctuated labeling of the receptor cells was made by vva 
 and bsl - i ( fig . 2c and 2c ) . the olfactory 
 receptor cells were not labeled by nine of the lectins used : bandeiraea 
 simplicifolia lectin - ii ( bsl - ii ) , dolichos biflorus agglutinin 
 ( dba ) , sophora japonica agglutinin ( sja ) , jacalin , peanut agglutinin ( pna ) , 
 erythrina cristagalli lectin ( ecl ) , ulex europaeus 
 agglutinin - i ( uea - i ) , lens culinaris agglutinin ( lca ) and phaseolus 
 vulgaris agglutinin - l ( pha - l ) ( fig . olfactory mucosa reacted with six lectins : lel ( a ) , ( a ) , stl ( b ) , ( b ) , vva ( c ) , 
 ( c ) , lca ( d ) , ( d ) , pna ( e ) , ( e ) and pha - l ( f ) and ( f ) . eight of the lectins used , wga , lel , stl , dsl , rca-120 , con a , pha - e and pha - l , labeled 
 both the olfactory supporting and basal cells with varying intensity . the localizations of 
 staining reactions were similar to each other and were restricted to the cytoplasm of both 
 the olfactory supporting and basal cells ( fig . vva labeled the cytoplasm of the basal cells ( fig . 2c and 2c ) . the free border of the olfactory epithelium was stained by 13 lectins : wga , s - wga , lel , 
 stl , dsl , sba , bsl - i , rca-120 , pna , ecl , con a , pha - e and pha - l with varying intensity . among the 21 lectins used , 13 lectins labeled bowman s glands with varying intensity : wga , 
 s - wga , stl , dsl , bsl - i , vva , rca-120 , jacalin , pna , ecl , con a , pha - e and pha - l . seven 
 lectins homogenously labeled the cytoplasm of bowman s gland cells : s - wga , stl , dsl , 
 rca-120 , jacalin , pna and ecl ( fig . bsl - i and vva had punctuated staining pattern , and the staining reactions were scattered 
 within the cytoplasm of the glandular cells ( fig . 2c ) . on the other hand , the staining patterns for wga , con a , pha - e and pha - l were 
 similar to each other , where they intensely stained the basal part of the glandular cells 
 ( fig . the lectin binding patterns in the olfactory mucosa are summarized in table 2table 2.lectin binding patterns of the sheep olfactory mucosalectinfree borderreceptor cellssupporting cellsbasal cellsbowman s glandswga++++++s - wga++/+/lel++++++++stl+++++++dsl++++++/bsl - iidbasba++/bsl - i+/++vva+++sjarca-120++++++jacalin+/pna+/+ecl++/uea - icon a++++++psa+/lca-pha - e++++++pha - l++++++ : negative staining , + / : faint staining , + : moderate staining , + + : intense 
 staining ..  : negative staining , + / : faint staining , + : moderate staining , + + : intense 
 staining . topographical and histological features of the respiratory mucosa : the 
 respiratory mucosa was located mostly in the middle part of the nasal cavity of the sheep . it lined the lateral wall of the nasal cavity and nasal septum and covered the vomeronasal 
 organ laterally . the respiratory mucosa was composed of respiratory epithelium and 
 connective tissue lamina propria ( fig . 3afig . ( a ) transverse section of the 
 respiratory mucosa containing both the respiratory epithelium and the underlying 
 lamina propria . bars=20 m . a , muscular artery ; g , goblet cell ; ng , nasal 
 glands ; v , venous sinus . ) . each type of cell had properties 
 that have been already described for the respiratory epithelial cells in sheep [ 20 , 22 ] . the 
 goblet cells consisted of an apical part containing mucopolysaccharide ( mucin ) granules and 
 a basal part containing the nucleus . the goblet cells reacted positively with both pas and 
 alcian blue ph 2.5 with varying intensity according to their mucin content ( fig . 3b and 3c ) . the number of the goblet cells of the 
 respiratory epithelium varied from one part to another in the nasal cavity . their nuclei were darkly stained 
 by hematoxylin and were smaller in size than those of the ciliated cells . their cytoplasm was only 
 faintly stained by eosin , and their nuclei were also paler than those of the basal and 
 goblet cells . ( a ) transverse section of the 
 respiratory mucosa containing both the respiratory epithelium and the underlying 
 lamina propria . bars=20 m . a , muscular artery ; g , goblet cell ; ng , nasal 
 glands ; v , venous sinus . within the propria , numerous glands with their ducts were found . 4.histological features of the serous and mixed glands within the lamina propria of the 
 respiratory mucosa . the mixed end - pieces comprised of both mucous , high cuboidal cells ( flattened 
 nuclei ) together with a few number of peripherally located serous cells surrounding a 
 relatively wide lumen ( fig . 4b , 4d and 4e ) . on the 
 other hand , the serous end - pieces consisted of pyramidal cells surrounding a narrow lumen . their nuclei were rounded and located at the basal portion of the cell ( fig . 4c , 4f and 4 g ) . both glandular types reacted positively , but in 
 a variable manner with pas and alcian blue ph 2.5 ( fig . mixed glands were stained with alcian blue ph 1.0 , but the 
 serous glands were not ( fig . 4d and 4f ) . histological features of the serous and mixed glands within the lamina propria of the 
 respiratory mucosa . lectin binding patterns in the respiratory mucosa : twelve out of the 21 
 lectins used in this study : wga , s - wga , lel , stl , bsl - ii , dba , sba , bsl - i , vva , rca-120 , ecl 
 and uea - i stained the goblet cells of the respiratory epithelium with varying intensity . the 
 staining patterns were similar to each other , and the reactions were localized to the mucin 
 granules in the apical cytoplasm . the intensity of staining was dependent on the amount of 
 mucin granules in each goblet cell ( fig . 5.respiratory mucosa reacted with 4 lectins : wga ( a ) , ( a ) lel ( b ) , ( b ) , dba ( c ) , ( c ) 
 and pha - l ( d ) , ( d ) . d , glandular duct ; g , goblet 
 cell ; m , mucous cell ; s , serous cell . bar=50 m in ( a)(d ) and 30 
 m in ( a)(d). , 5b and 5b ) . respiratory mucosa reacted with 4 lectins : wga ( a ) , ( a ) lel ( b ) , ( b ) , dba ( c ) , ( c ) 
 and pha - l ( d ) , ( d ) . d , glandular duct ; g , goblet 
 cell ; m , mucous cell ; s , serous cell . bar=50 staining patterns for dba , sba and vva in the respiratory epithelium were similar to each 
 other . there were alternative intervals of stained and unstained regions in the basal and 
 ciliated cells of the respiratory epithelium ( fig . 5c and 
 5c ) . eight lectins : dsl , dba , sba , vva , rca-120 , uea - i , con a and pha - l , labeled the ciliated 
 cells with varying intensity ( fig . the staining patterns were similar to each other ; these lectins labeled both 
 their cell membranes and cytoplasm . the basal cells were stained by 11 lectins : lel , stl , dsl , dba , sba , bsl - i , vva , uea - i , con 
 a , pha - e and pha - l with varying intensity . cell membranes and cytoplasm of the basal cells 
 were stained , but their nuclei were not stained ( fig . the free border of the respiratory epithelium was labeled by 15 lectins : wga , s - wga , lel , 
 stl , dsl , dba , sba , bsl - i , vva , rca-120 , ecl , uea - i , con a , pha - e and pha - l with varying 
 intensity . four lectins out of the 21 lectins used : sja , jacalin , pna and lca were negative in neither 
 epithelium . several spindle - shaped cells of unknown origin with dark nuclei and scanty cytoplasm were 
 detected within the respiratory epithelium mostly located close to the goblet cells ( fig . these cells were labeled with varying intensity by 7 lectins : lel , dba , sba , bsl - i , 
 vva , uea - i and pha - l . the staining reactions were similar to each other , including both 
 their cytoplasm and nuclei ( fig . 6b ) . only uea - i 
 distinguished their cytoplasm from nuclei by staining the cytoplasm intensely with the 
 nuclei remaining unstained ( fig . the lectin binding patterns in the respiratory epithelium are summarized in table 3table 3.lectin binding patterns of the sheep respiratory epitheliumlectinfree bordergoblet cellsbasal cellsciliated cellsunrecognized type of cellswga+++s - wga+++lel+++/+/++stl+++/dsl++++bsl - ii+dba+*+/++++*++sba+*++++++bsl - i+/+++vva+*++++sjarca-120+++/+jacalinpnaecl++uea - i+/++++/++con a+++/psalcapha - e++/pha - l++++/+ : negative staining , + / : faint staining , + : moderate staining , + + : intense 
 staining . * : remarkable staining ( binding reaction is not equally distributed along 
 the cell membrane ) ..  : negative staining , + / : faint staining , + : moderate staining , + + : intense 
 staining . * : remarkable staining ( binding reaction is not equally distributed along 
 the cell membrane ) . in the lamina propria , the mucous cells were stained intensely by 9 among the 21 lectins 
 used in this study : wga , s - wga , stl , dba , sba , vva , ecl , uea - i and pha - e ( fig . 5a and 5c ) . the serous glands as well as the 
 serous cells of the mixed glands were labeled by six lectins : lel , dsl , bsl - ii , con a , pha - e 
 and pha - l with varying intensity ( fig . 5b and 
 5d ) . the lectin binding patterns of the nasal glands are summarized in table 4table 4.lectin binding patterns of the glands in the lamina propria of the respiratory 
 epitheliumlectinserous glandsmixed glandsserous cellsmucous cellswga++s - wga++lel++++stl+dsl++bsl - ii+/+/dba++sba+bsl - ivva++sjarca-120jacalinpnaecl++uea - i++con a++psalcapha - e+++pha - l++++ : negative staining , + / : faint staining , + : moderate staining , + + : intense 
 staining ..  : negative staining , + / : faint staining , + : moderate staining , + + : intense 
 staining . in the present study , we have revealed the details of the glycoconjugate localizations in 
 sheep . our histological and histochemical studies using h&e , pas and alcian blue had 
 good agreement with previous reports by kavoi et al . , khamas and ghoshal   and 
 taher et al . . on the contrary , 
 some of our findings using lectin histochemistry were inconsistent with the previous report 
 in the olfactory epithelium by scocco et al . . our study showed eight lectins labeled both the supporting and basal 
 cells with varying intensity : wga , lel , stl , dsl , rca-120 , con a , pha - e and pha - l . on the 
 other hand , scocco et al . labeled these cells using nine lectins and found that six lectins : wga , con a , lta , uea - i , 
 sba and gsa - ib4 were positive in these cells . only 2 lectins , wga and con a , were positive 
 in both of our work and scocco s work . in the respiratory epithelium , the case was the same 
 as in the olfactory epithelium . our results and scocco s results showed only partial 
 consistency ; 5 of mutually examined lectins ( con a , dba , sba , uea - i and rca ) were positive 
 by both studies , but one lectin ( wga ) was positive only in scocco s study . the discrepancies 
 in the results between scocco et al .   and our own study may at least be partially due to the differences in the 
 fixatives employed ( carnoy s fluid for 24 hr at room temperature and postfixed in 2% calcium 
 acetate-4% paraformaldehyde solution 1:1 by scocco et al . ) . but , it could 
 also be attributed to the differences between the corriedale and lacaune sheep breeds . twelve lectins stained both the free border of the olfactory epithelium and that of the 
 respiratory epithelium : wga , s - wga , lel , stl , dsl , sba , bsl - i , rca-120 , ecl , con a , pha - e 
 and pha - l , indicating that the expression pattern of glycoconjugate was similar between the 
 free border of these two epithelia . the lectin stainings in the free border may come 
 partially from the glycoconjugates in the mucus covering the surface of the epithelium and 
 partially from those expressed in the cell membrane of the cilia or the microvilli of the 
 goblet , ciliated , receptor or supporting cells . the mucus at the surface of the olfactory 
 mucosa constitutes the milieu in which perireceptor events associated with olfactory signal 
 transduction occur . it is tempting to speculate , 
 firstly , that the goblet cells and mucous nasal glands in the respiratory epithelium may 
 contribute to the function of the olfactory epithelium in the perception of odorants in the 
 olfactory receptor cells . the secretory products of the respiratory epithelium may 
 distribute caudally on the free border of the olfactory epithelium   by the movement of cilia and gravity and thus accelerating the 
 movement of various odorants towards their targeted olfactory receptor cells , resulting in 
 enhanced perception of odorants in the olfactory epithelium . another possibility is that the 
 secretory products of the bowman s glands may distribute cranially on the free border of the 
 respiratory epithelium and thus help in discarding of dust particles and foreign organisms 
 from the respiratory epithelium , accelerating the function of the goblet cells as a part of 
 the nasal cavity immune system . the 2nd idea is that there are similarities in the 
 carbohydrate expressions of the cell membranes of the two epithelia . although it is possible 
 that the secretion from bowman s and nasal glands affects the lectin - binding patterns on the 
 free border , the patterns may be attributed to the sugar residues expressed on the olfactory 
 knobs , cilia or microvilli of the free borders , since some of lectins binding to the free 
 borders did not bind to the associated glands . for example , lel stained the free borders of 
 both the olfactory and respiratory epithelia , but its staining intensity in the bowman s 
 glands and goblet cells is faint . this may indicate that the sugar residues that were 
 labeled by lel may originate from the cell processes and not secreted or expressed either in 
 the bowman s glands or goblet cells . these cells 
 were labeled by seven out of the 21 lectins used in this study . exclusively , uea - i stained 
 the cytoplasm of these cells , but not the nuclei . as far as we know , this is the first 
 report detecting this type of cell in the respiratory epithelium of sheep . we speculate that 
 the presence of these cells can be attributed to one of the different cell cycle phases of 
 the goblet or ciliated cells , as immature differentiated cells .
abstractthe olfactory and respiratory mucosae of the corriedale sheep were examined 
 using lectin histochemistry in order to clarify the histochemical and glycohistochemical 
 differences between these two tissues . 
 the olfactory epithelium was stained with 13 
 lectins out of 21 lectins examined , while the respiratory epithelium was positive to 16 
 lectins . 
 the free border of both of the olfactory and respiratory epithelia was stained 
 with 12 lectins : wheat germ agglutinin ( wga ) , succinylated - wheat germ agglutinin ( s - wga ) , 
 lycopersicon esculentum lectin ( lel ) , solanum 
 tuberosum lectin ( stl ) , datura stramonium lectin ( dsl ) , 
 soybean agglutinin ( sba ) , bandeiraea simplicifolia lectin - i ( bsl - i ) , 
 ricinus communis agglutinin - i ( rca-120 ) , erythrina 
 cristagalli lectin ( ecl ) , concanavalin a ( con a ) , phaseolus 
 vulgaris agglutinin - e ( pha - e ) and phaseolus vulgaris 
 agglutinin - l ( pha - l ) . the associated glands of the olfactory mucosa , bowman s glands , were 
 stained with 13 lectins . while both the goblet cells and mucous nasal glands were stained 
 with 8 lectins 
 ; five of them ( wga , s - wga , stl , vicia villosa agglutinin 
 ( vva ) and ecl ) were mutually positive among the bowman s glands , mucous nasal glands and 
 the goblet cells . 
 these findings indicate that the glycohistochemical characteristics of 
 the free borders of both olfactory and respiratory epithelia are similar to each other , 
 suggesting that secretions from the bowman s glands and those of the goblet cells and 
 mucous nasal glands are partially exchanged between the surface of two epithelia to 
 contribute the functions of the respiratory epithelium and the olfactory receptor cells , 
 respectively .