adult soft - shelled turtles ( 2 males and 4 females ) were purchased from a local supplier 
 tobun ( kamikita , japan ) .
all procedures were done in accordance with the guideline for care 
 and use of animal experiments and approved by the animal care and use committee at iwate 
 university .
the animals were anesthetized by intraperitoneal injection of sodium 
 pentobarbital ( 64.8 mg / kg ) .
two animals ( 1 male and 1 female ) were perfused transcardially 
 with ringer s solution followed by bouin s solution without acetic acid .
the upper jaw was 
 dissected , immersed in the same fixative solution overnight at 4c and decalcified in 10% 
 ethylenediamine tetra - acetic acid ( edta ) in 0.1 m phosphate buffer at 4c for two weeks .
the 
 specimens were routinely embedded in paraffin and cut at 57 m thick . 

sections were stained with pas or ab ( ph 1.0 or ph 2.5 ) , or processed for lectin 
 histochemistry as described previously . for transmission electron microscopy , samples were collected from four animals ( 1 male and 
 3 females ) and processed as described previously .
the nasal cavity of soft - shelled turtles was divided into the upper and lower chambers 
 ( fig . 1a and 1bfig . 1.the olfactory organs in soft - shelled turtle .
uc , upper chamber ; lc , lower chamber ; on , olfactory nerve ; ob , olfactory 
 bulb . coronary section at line b is shown in b. ( b ) the right nasal cavity .
( c ) he stained sections showing upper 
 chamber epithelium ( upper left ) , the bowman s glands ( lower left , bg ) and the lower 
 chamber epithelium ( right ) in the nasal cavity of soft - shelled turtles .
the nuclei of 
 supporting cells ( sp ) , receptor cells ( rc ) and basal cells ( bc ) were situated in the 
 apical , intermediate and basal layers of the upper and lower chamber epithelia .
( d ) 
 pas , alcian blue ( ab ) ph 1.0 and ab ph 2.5 stainings in the upper chamber epithelium 
 ( top panels ) , lower chamber epithelium ( middle panels ) and the bowman s glands ( bottom 
 panels ) .
the apical portion of the upper chamber epithelium was stained with pas , ab 
 ph 1.0 and ab ph 2.5 ( bidirectional arrows ) . in the lower chamber epithelium , 
 cytoplasm near the free border ( arrowheads ) was more intensely stained than the 
 supranuclear region of the supporting cells ( bidirectional arrows ) with pas and ab ph 
 2.5 .
the bowman s glands were 
 positive for pas , but negative for ab ph 1.0 and ab ph 2.5 .
the nuclei were 
 counterstained with nuclear fast red in the sections stained with ab ph 1.0 .
scale 
 bars : 50 m . ) . both of the upper and lower chamber epithelia consist of three types of 
 cells , the supporting cells , receptor cells and basal cells ( fig .
nuclei of the supporting , receptor and basal 
 cells were situated in the apical , middle and basal layers of the epithelium , respectively . 

the associated glands ( the bowman s glands ) were observed in the upper chamber epithelium , 
 but not in the lower chamber epithelium ( fig . 

in addition , staining patterns for pas and ab ph 2.5 differed between the two 
 epithelia ( fig .
pas and ab ph 2.5 intensely 
 stained the apical one - third of the upper chamber epithelium .
in contrast , they stained the 
 apical portion near the free border intensely and the supranuclear region of supporting 
 cells less intensely in the lower chamber epithelium .
moreover , the upper chamber epithelium 
 and the lower chamber epithelium were differently stained by ab ph 1.0 : the upper chamber 
 epithelium was positive for ab ph 1.0 , but the lower chamber epithelium was negative .
the 
 bowman s glands were positive for pas , but negative for ab ph 1.0 and ab ph 2.5 .
uc , upper chamber ; lc , lower chamber ; on , olfactory nerve ; ob , olfactory 
 bulb . coronary section at line b is shown in b. ( b ) the right nasal cavity .
( c ) he stained sections showing upper 
 chamber epithelium ( upper left ) , the bowman s glands ( lower left , bg ) and the lower 
 chamber epithelium ( right ) in the nasal cavity of soft - shelled turtles .
the nuclei of 
 supporting cells ( sp ) , receptor cells ( rc ) and basal cells ( bc ) were situated in the 
 apical , intermediate and basal layers of the upper and lower chamber epithelia .
( d ) 
 pas , alcian blue ( ab ) ph 1.0 and ab ph 2.5 stainings in the upper chamber epithelium 
 ( top panels ) , lower chamber epithelium ( middle panels ) and the bowman s glands ( bottom 
 panels ) .
the apical portion of the upper chamber epithelium was stained with pas , ab 
 ph 1.0 and ab ph 2.5 ( bidirectional arrows ) .
in the lower chamber epithelium , 
 cytoplasm near the free border ( arrowheads ) was more intensely stained than the 
 supranuclear region of the supporting cells ( bidirectional arrows ) with pas and ab ph 
 2.5 .
the bowman s glands were 
 positive for pas , but negative for ab ph 1.0 and ab ph 2.5 .
the nuclei were 
 counterstained with nuclear fast red in the sections stained with ab ph 1.0 .
scale 
 bars : 50 m . in the apical part of the upper chamber epithelium , the dendrites of the receptor cells 
 bearing cilia on the tip ( fig .
( a ) secretory granules filled the supranuclear region of the supporting cells ( sp ) 
 in the upper chamber epithelium .
( b ) most secretory granules were distributed near 
 the free border of supporting cells in the lower chamber epithelium .
arrows in a and 
 b indicate receptor cells bearing cilia on the tip of the dendrites .
( c ) secretory 
 granules of the supporting cells in the upper chamber epithelium , 12 
 m in diameter , consisted of the marginal part of moderate 
 density and central part of low density .
some of the secretory granules contained a 
 core of high density ( arrowheads ) .
( d ) secretory granules of the supporting cells in 
 the lower chamber epithelium , 0.51 m in diameter , consisted of 
 two parts of moderate and high density .
( e ) the glandular cells in the bowman s 
 glands contained secretory granules of high density .
scale bars : 2 
 m in a and b , 0.5 m in c  e .
, arrows ) alternated with the cytoplasm of supporting cells bearing 
 microvilli on the free border ( fig .
the secretory 
 granules of supporting cells in the upper chamber epithelium , measuring 12 
 m in diameter , showed a bipartite structure with the marginal part 
 displaying moderate density and central part displaying low density ( fig . 2c ) .
in addition , a core of high density was observed in some 
 secretory granules ( fig . 2c , arrowheads ) . in the 
 apical part of lower chamber epithelium , the dendrites of receptor cells bearing cilia 
 alternated with the cytoplasm of supporting cells bearing microvilli ( fig .
most secretory granules were distributed near the 
 free border of supporting cells in the lower chamber epithelium ( fig .
the secretory granules of supporting cells , 0.51 
 m in diameter , showed a bipartite structure of moderate and high 
 densities ( fig .
secretory granules of high 
 density , approximately 1 m in diameter , were abundant in the glandular 
 cells of the bowman s gland ( fig .
( a ) secretory granules filled the supranuclear region of the supporting cells ( sp ) 
 in the upper chamber epithelium .
( b ) most secretory granules were distributed near 
 the free border of supporting cells in the lower chamber epithelium .
arrows in a and 
 b indicate receptor cells bearing cilia on the tip of the dendrites .
( c ) secretory 
 granules of the supporting cells in the upper chamber epithelium , 12 
 m in diameter , consisted of the marginal part of moderate 
 density and central part of low density .
some of the secretory granules contained a 
 core of high density ( arrowheads ) .
( d ) secretory granules of the supporting cells in 
 the lower chamber epithelium , 0.51 m in diameter , consisted of 
 two parts of moderate and high density .
( e ) the glandular cells in the bowman s 
 glands contained secretory granules of high density .
scale bars : 2 
 m in a and b , 0.5 m in c  e . cellular expression of carbohydrate chains
staining patterns for lectins were evaluated in each region where the secretory 
 granules were observed by transmission electron microscopy : the apical one - third of the 
 upper chamber epithelium ( fig . 3afig .
( a ) the apical 
 portions of the upper chamber epithelium ( top panels ) and lower chamber epithelium 
 ( bottom panels ) .
bidirectional arrows indicate the supranuclear region , and arrowheads 
 indicate the cytoplasmic region near the free border of the supporting cells .
( left 
 panels ) uea - i stained the supporting cells in the lower chamber epithelium more 
 intensely than those in the upper chamber epithelium .
( middle panels ) rca120 equally 
 stained the supporting cells in the upper chamber epithelium and those in the lower 
 chamber epithelium .
( right panels ) lel stained the supporting cells in the upper 
 chamber epithelium more intensely than those in the lower chamber epithelium .
( b ) 
 bowman s glands were stained intensely by swga , moderately by vva and weakly by pna . 

scale bars : 25 m . , top panels ) , the superficial portion ( fig . 

3a , bottom panels , bidirectional arrows ) of the lower chamber 
 epithelium , and the glandular cells of the bowman s gland ( fig .
eighteen lectins stained the secretory granules of 
 supporting cells in the upper and/or lower chamber epithelium ( table 1table 1.lectin binding patterns in the supporting cells of the upper and lower 
 chamber epithelia and the bowman s glandslectin ( abbreviation)upper chamber epitheliumlower chamber epitheliumbowman s glandswheat germ agglutinin ( wga)+++++succinylated wheat germ agglutinin ( s - wga)+/+/++lycopersicon esculentum lectin ( lel)++++solanum tuberosum lectin ( stl)++/+datura stramonium lectin ( dsl)++++bandeiraea simplicifolia lectin - ii ( bsl - ii)++dolichos biflous agglutinin ( dba)+++soybean agglutinin ( sba)+/++bandeiraea simplicifolia lectin - i ( bsl - i)+/+/vicia villosa agglutinin ( vva)++++sophora japonica agglutinin ( sja)ricinus communis agglutinin - i ( rca120)+++++jacalin++/peanut agglutinin ( pna)+++/erythrina cristagalli lectin ( ecl)+++ulex europaeus agglutinin - i ( uea - i)+/+++concanavalin a ( con a)++++++pisum satibum agglutinin ( psa)++lens culinaris agglutinin ( lca)++phaseolus vulgaris agglutinin - e ( pha - e)+++phaseolus vulgaris agglutinin - l ( pha - l)+/ , negative staining ; + / , faint staining ; + , moderate staining ; + + , intense 
 staining . ) . among them , 14 lectins stained the supporting cells in the upper chamber 
 epithelium and those in the lower chamber epithelium differently .
eleven lectins , including 
 dsl , dba , sba , bsl - i , vva , jacalin , pna , uea - i , psa , lca and pha - e , stained the supporting 
 cells in the lower chamber epithelium more intensely than those in the upper chamber 
 epithelium ( fig .
four lectins , 
 including swga , rca120 , ecl and cona , stained the supporting cells in the upper chamber 
 epithelium and those in the lower chamber epithelium equally ( fig .
three lectins , including wga , lel and stl , stained 
 the supporting cells in the upper chamber epithelium more intensely than those in the lower 
 chamber epithelium ( fig .
twenty 
 lectins , excluding sja , stained the secretory granules in the glandular cells of the 
 bowman s glands ( table 1 ) .
five lectins , 
 including wga , swga , bsl - ii , dba and cona , stained the bowman s glands intensely .
eleven 
 lectins , including lel , stl , dsl , sba , vva , rca120 , ecl , uea - i , psa , lca and pha - e , stained 
 moderately , and 4 lectins , including bsl - i , jacalin , pna and pha - l , stained weakly ( fig .
fourteen lectins stained 
 the supporting cells of upper chamber epithelium and the bowman s glands differently ( table 1 ) .
( a ) the apical 
 portions of the upper chamber epithelium ( top panels ) and lower chamber epithelium 
 ( bottom panels ) .
bidirectional arrows indicate the supranuclear region , and arrowheads 
 indicate the cytoplasmic region near the free border of the supporting cells .
( left 
 panels ) uea - i stained the supporting cells in the lower chamber epithelium more 
 intensely than those in the upper chamber epithelium .
( middle panels ) rca120 equally 
 stained the supporting cells in the upper chamber epithelium and those in the lower 
 chamber epithelium .
( right panels ) lel stained the supporting cells in the upper 
 chamber epithelium more intensely than those in the lower chamber epithelium .
( b ) 
 bowman s glands were stained intensely by swga , moderately by vva and weakly by pna . 

 , negative staining ; + / , faint staining ; + , moderate staining ; + + , intense 
 staining .
as we demonstrated in this study , the supporting cells contained secretory granules both in 
 the upper chamber epithelium and lower chamber epithelium in the nasal cavity of 
 soft - shelled turtles .
supporting cells of the upper chamber epithelium contained secretory 
 granules more abundantly than those of the lower chamber epithelium : the secretory granules 
 filled the supranuclear region of the supporting cells in the upper chamber epithelium : 
 whereas , most of the secretory granules were localized near the free border and were 
 scarcely distributed in the supranuclear region of the supporting cells in the lower chamber 
 epithelium . in addition , the upper chamber epithelium possessed the associated glands , but 
 the lower chamber epithelium did not . these evidences
suggest variable lubrication ability 
 between the upper and lower chamber epithelia : secretory activity is higher in the upper 
 chamber epithelium than in the lower chamber epithelium . in squamates ,
the presence or 
 absence of associated glands and the amount of the secretory granules are different between 
 the oe and the vne [ 21 , 27 ] .
for example , the oes of snakes , gekkos and scincomorpha associate 
 with glands and contain abundant secretory granules in the supporting cells : whereas , their 
 vnes lack the associated glands and contain few secretory granules in the supporting cells 
 [ 25 , 26 ] . 

the lumen of squamate vno is always filled with fluids , which have both an intrinsic source 
 and , at least partially , an extrinsic source ( most likely the harderian glands ) . in the case 
 of soft - shelled turtles ,
the surface of the lower chamber epithelium is always in contact 
 with extrinsic water , possibly leading to the lower secretory activity in the lower chamber 
 epithelium as compared with the upper chamber epithelium . in general
, the secretory products of exocrine cells are classified into 3 types : serous 
 type containing neutral mucopolysaccharides , mucous type containing acid mucopolysaccharides 
 and seromucous type containing both neutral and acid mucopolysaccarides .
the secretory granules in the bowman s glands of 
 soft - shelled turtles have been suggested to be serous , because they were pas - positive and 
 ab - negative and showed high electron - density .
the bowman s glands , which present in all 
 vertebrate species except fish and some amphibians , are generally serous in amphibians , 
 reptiles and birds . also , in the soft - shelled 
 turtles , the bowman s glands contain serous secretory granules , suggesting that their 
 secretory products play a significant role shared among non - mammalian bowman s glands .
on 
 the other hand , the supporting cells of both upper chamber epithelium and lower chamber 
 epithelium were positive for pas and ab ph 2.5 , suggesting that they contain mucous or 
 seromucous secretory granules .
by the electron microscopic observation , the supporting cells 
 of both chamber epithelia have been demonstrated to contain secretory granules of 
 heterogeneous electron - density , suggesting that they are seromucous .
furthermore , supporting 
 cells of upper chamber epithelium and lower chamber epithelium were suggested to contain 
 secretory granules of different compositions , because they exhibited inconsistent staining 
 for ab ph 1.0 . in order to make further confirmation for the differences in the properties of secretory 
 granules
, we analyzed the expression of carbohydrate chains by lectin histochemistry in the 
 area where secretory granules were distributed , and compared them between supporting cells 
 of upper chamber epithelium and those of the lower chamber epithelium .
although 4 lectins 
 stained the supporting cells of both chamber epithelia equally , 14 lectins stained the 
 supporting cells of either upper or lower chamber epithelium more intensely . among them , 3 
 lectins stained the supporting cells of upper chamber epithelium more intensely than those 
 of the lower chamber epithelium , and 11 lectins stained the supporting cells of lower 
 chamber epithelium more intensely than those of the upper chamber epithelium .
although 
 electron - microscopic data indicated that the amount of the secretory granules in the 
 supporting cells of lower chamber epithelium is apparently smaller than those of the upper 
 chamber epithelium , most lectins stained the supporting cells of lower chamber epithelium 
 more intensely than those of the upper chamber epithelium .
thus , it seemed that the 
 differences in the lectin staining between them are not due to the differences in the amount 
 of secretory granules but instead due to the differences in their carbohydrate expressions , 
 i.e. the differences in the properties of secretory products .
in addition to the differences in staining patterns for pas and ab or in the 
 electron - density , the secretory granules in the bowman s glands showed different binding 
 patterns to lectins from the patterns of the supporting cells in the upper and lower chamber 
 epithelia .
for example , supporting cells of both epithelia were negative for bsl - ii and 
 pha - l , whereas the bowman s glands were positive .
the supporting cells of both epithelia 
 were weakly positive for s - wga , whereas the bowman s glands were intensely positive .
these 
 evidences suggest that the properties of secretory granules are different among them . in this study
, we revealed that the associated glands are present only in the upper chamber 
 epithelium , that the supporting cells of upper chamber epithelium contain secretory granules 
 more abundantly than those of the lower chamber epithelium and that the properties of 
 secretory granules in the supporting cells are different between the upper and lower chamber 
 epithelia in the nasal cavity of soft - shelled turtles .
the fluid layer covering the surface 
 of olfactory sensory epithelium plays important roles in olfaction as mentioned above , and 
 the properties of fluid layer differ among functionally distinct epithelia .
considering the 
 lubricating ability as one of the indices for olfactory functions , differences in the 
 olfactory function are suggested by the differences in the amount and properties of 
 secretory granules between the upper and lower chamber epithelia in the olfactory organ of 
 soft - shelled turtles . in general ,
semi - aquatic turtles show sniffing behavior under water as 
 well as on land .
even when turtles are under water and the lower chamber is filled with 
 water , the upper chamber could be filled with air owing to the structure of nasal cavity . 

thus , in the nasal cavity of semi - aquatic turtles , the upper and lower chamber epithelia 
 could be considered as the olfactory organs that function in the air and under water , 
 respectively [ 5 , 28 ] .
also , in some amphibians , the olfactory organ that functions in the air is 
 different from the olfactory organ that functions under water in the amount and properties 
 of secretory granules .
two distinct sensory epithelia present in the main olfactory system 
 of adult xenopus laevis , the oe ( functioning in the air ) and the middle 
 chamber epithelium ( functioning under water ) , are such examples [ 9 , 10 ] : the bowman s glands are 
 present , and the supporting cells contain abundant secretory granules in the oe ; whereas , 
 associated glands are not present , and the supporting cells contain few secretory granules 
 in the middle chamber epithelium [ 14 , 24 ] .
the xenopus oe functions under 
 water in the larval stage and functions in the air during adulthood . during metamorphosis , 

the bowman s glands are formed , the amount and size of the secretory granules increase , and 
 their electron - density and lectin - binding patterns change in the supporting cells of the oe 
 [ 6 , 14 , 33 ] .
also , in the olfactory organs of soft - shelled 
 turtles , the upper and lower chamber epithelia seem to be the olfactory organs that function 
 in the air and under water , respectively .
 in general , the nasal cavity of turtles is divided into two chambers : the upper chamber , 
 lined with the olfactory epithelium containing ciliated olfactory receptor cells , and the 
 lower chamber , lined with the vomeronasal epithelium containing microvillous receptor 
 cells . in the nasal cavity of soft - shelled turtles , however , differences between the upper 
 and lower chamber epithelia are unclear due to the presence of ciliated receptor cells in 
 both epithelia . in the olfactory organ of vertebrates , 
 the surface of sensory epithelium 
 is covered with secretory products of associated glands and supporting cells , playing 
 important roles in the olfaction by dissolving odorants and transporting them to the 
 olfactory receptors . here , 
 the associated glands and supporting cells in the olfactory 
 organ of soft - shelled turtles were analyzed histochemically and ultrastructurally . 
 the 
 upper chamber epithelium possessed associated glands , constituted by cells containing 
 serous secretory granules ; whereas , the lower chamber epithelium did not . in the upper 
 chamber epithelium 
 , secretory granules filled the supranuclear region of supporting cells , 
 while most of the granules were distributed near the free border of supporting cells in 
 the lower chamber epithelium . 
 the secretory granules in the supporting cells of both 
 epithelia were seromucous , but alcian blue stained them differently from each other . in 
 addition , distinct expression of carbohydrates was suggested by the differences in lectin 
 binding . 
 these data indicate the quantitative and qualitative differences in the secretory 
 properties between the upper and lower chamber epithelia , suggesting their distinct roles 
 in the olfaction . 