experimental birds : white leghorn chicks ( n=12 , male ) at one day of age 
 were commercially obtained from komatsu shukeijyo ( matsumoto , japan ) and reared in our 
 laboratory given a commercial diet and water ad libitum and under 
 controlled light condition ( 12 hr light : 12 hr darkness ) up to one week of age .
chicks were 
 treated in accordance with the  guideline for regulation of animal experimentation ( 1997 )  
 of faculty of agriculture , shinshu university .
tissue samples : chicks were perfused with a mixture of 4% paraformaldehyde 
 and 0.01% glutaraldehyde in phosphate buffer ( ph7.6 ) following 0.75% nacl solution under 
 anesthesia with diethyl ether . proximal and distal portions of ileum were immediately 
 dissected out as the tissue samples and then immersed in the same perfusate for 5 hr at 4c . 

tissue samples were cut into small blocks and pieces and embedded in paraffin wax and epoxy 
 resin , quetol 812 , respectively according to each standard procedure .
immunofluorescent technique : paraffin sections cut at 5 
 m thickness were treated with 2.5% normal donkey serum ( ihr-8135 , 
 immunobioscience , mukilteo , wa , u.s.a . ) for 20 min and incubated with the mouse monoclonal 
 antibody against synthetic human glp-1 ( aa 736 amide ) ( diluted to 1:2,000 , a6104.1 , 
 immunodiagnostik , bensheim , germany ) for 24 hr .
after several washing with phosphate 
 buffered saline ( pbs ) , paraffin sections were subsequently incubated with rabbit anti - human 
 ( arg)-glp-2 serum ( 1:500 , h-028 - 14 , phenix pharmaceuticals , burlingame , ca , 
 u.s.a . ) for 24 hr .
incubation of sections with the cocktail of fitc - labeled donkey 
 anti - mouse ig g ( 1:100 , gtx85337 , genetex , irvine , ca , u.s.a . ) and rhodamine - labeled donkey 
 anti - rabbit ig g
( 1:100 , 611 - 700 - 127 , rockland immunochemicals , gillbertsville , pa , u.s.a . ) 

thermo fisher scientific , fremont , ca , u.s.a . ) , observed and photomicrographed 
 under a fluorescent microscope ( axioimager , zeiss , gttingen , germany ) .
the specificities of primary antibodies used in this study were documented by the 
 manufacturers and in previous reports [ 6 , 19 ] .
primary antibodies against glp-1 and glp-2 mentioned 
 above were preabsorbed with chicken / common turkey glp-1(736 ) amide 
 ( 10m / ml , h-5824 , bachem , bubendorf , switzerland ) at 4c for 
 24 hr and then used for confirming the specificities of them .
preabsorbed glp-1 monoclonal 
 antibody showed the negative reaction , but preabsorbed glp-2 antiserum did the specific 
 immunoreactivity .
moreover , single immunofluorescent staining was carried out for each 
 primary antibody and compared with double immunofluorescent staining .
negative control 
 sections were incubated with the normal donkey serum instead of the specific primary 
 antibody or in the absence of the primary antibody .
paraffin sections from rat ileum were used for positive control sections and showed the 
 specific immunoreactivity for each primary antibody .
the specificity of the secondary 
 antiserum was controlled by the omission of the primary antibody .
immunocytochemistry : ultrathin sections were made with an ultramicrotome 
 ( super nova , reichert - jung , vienna , austria ) by using glass knives and mounted on nickel 
 grids .
ultrathin sections , pretreated with 1% sodium periodate solution and 1% normal donkey 
 serum subsequently , were incubated with the mouse monoclonal antibody against glp-1 
 ( 1:8,000 ) overnight .
after several washing with pbs , they were treated with colloidal gold 
 ( 12 nm in diameter)-labeled donkey anti - mouse ig g serum ( 1:200 , 715 - 205 - 150 , jackson immuno 
 research , west grove , pa , u.s.a . ) for 2 hr , stained with ti blue ( nisshin - em , tokyo , japan ) 
 and observed under a transmission electron microscope ( jem-1400 , jeol , tokyo , japan ) .
double immunocytochemical staining was applied for the detection of colocalization of glp-1 
 with glp-2 at electron microscopic level .
after incubation with the monoclonal antibody 
 against glp-1 , sections were subsequently incubated with rabbit anti - glp-2 serum ( 1:2,000 ) . 
 and
then , ultrathin sections were treated with a mixture of colloidal gold ( 12 nm in 
 diameter)-labeled donkey anti - mouse ig g serum mentioned above and colloidal gold ( 6 nm in 
 diameter)-labeled donkey anti - rabbit ig g serum ( 1:200 , 711 - 205 - 152 , jackson immuno 
 research ) for 2 hr , stained with ti blue and observed under a transmission electron 
 microscope .
the specificity of the colloidal gold - labeled secondary antiserum was controlled by the 
 omission of the primary antibody .
morphometry : morphometrical analysis was carried out to characterize 
 secretory granules of l - cells .
the shortest and longest diameters of secretory granules were 
 measured by using an image analyzer ( ks400 , carl zeiss , gttingen , germany ) . 

single immunofluorescent staining for glp-1 and glp-2 revealed that endocrine cells showing 
 immunoreactivity for each peptide were scattered in the villous epithelium and crypt of the 
 chicken ileum ( fig .
1.immunofluorescent staining for glp-1 ( a , c ) and glp-2 ( b , c ) in the chicken distal 
 ileum .
a , b : single immunofluorescent staining for glp-1 ( a ) and glp-2 ( b ) .
both 
 immunoreactive cells are scattered in villous epithelium and crypt of the distal ileum 
 and show the similar localization to that indicated by double immunofluorescent 
 staining .
c , c : double immunofluorescent staining for glp-1 ( c ) and glp-2 ( c ) .
both immunoreactive cells showed the similar localization in the ileum to that 
 indicated by double immunofluorescent staining as mentioned below and our previous 
 observation .
immunofluorescent staining for glp-1 ( a , c ) and glp-2 ( b , c ) in the chicken distal 
 ileum .
a , b : single immunofluorescent staining for glp-1 ( a ) and glp-2 ( b ) .
both 
 immunoreactive cells are scattered in villous epithelium and crypt of the distal ileum 
 and show the similar localization to that indicated by double immunofluorescent 
 staining .
c , c : double immunofluorescent staining for glp-1 ( c ) and glp-2 ( c ) .
bar=20 
 m . l - cells showing immunoreactivity for both glp-1 and glp-2 were observed in the whole ileum 
 ( figs . 1c , 
 1c and 2 ) .
they were located in 
 epithelium of crypts and lower part of intestinal villi and showed comma - like or flask - like 
 shape ( fig .
2.double immunofluorescent staining for glp-1 ( a ) and glp-2 ( b ) in villous epithelium 
 of the chicken distal ileum .
l - cells showing immunoreactivity for both glp-1 and glp-2 
 have a long cytoplasmic process ( small arrow ) and are in contact with intestinal lumen 
 ( large arrows ) .
l - cells showing immunoreactivity for only glp-1 were located in epithelium of lower and 
 middle parts of intestinal villi ( fig .
double immunofluorescent staining for glp-1 ( a ) and glp-2 ( b ) in villous epithelium 
 of the chicken distal ileum .
l - cells showing immunoreactivity for both glp-1 and glp-2 
 have a long cytoplasmic process ( small arrow ) and are in contact with intestinal lumen 
 ( large arrows ) .
, l - cells labeled with gold particles of 12 nm in diameter 
 were found in epithelium of crypts and lower and middle parts of intestinal villi ( fig .
( a ) low magnification 
 view of l - cell showing glp-1-immunoreactivity and located in villous epithelium .
bar=1 m . ( b ) 
 high magnification view of a secretory granule from l - cell .
particles of colloidal 
 gold ( 12 nm in diameter ) are diffusely arranged on a granule .
( c ) low magnification view of l - cells showing glp-1 and 
 -2-immunoreactivity and located in epithelium of crypt .
bar=1 m . ( d ) 
 high magnification view of a secretory granule from l - cell .
both sizes of particles of 
 colloidal gold ( 6 and 12 nm in diameter ) are diffusely arranged on a granule .
( e ) high 
 magnification view of secretory granules of ec cell in chicken small intestine .
gold particles were mainly found on secretory granules , but rarely on cytosol or 
 other organelles .
l - cells were flask - like or comma - like in shape ( fig .
they were covered apically 
 with microvilli , so - called open - type endocrine cells .
their secretory granules without halo 
 were accumulated mainly in the basal cytoplasm of l - cells .
they were round to oval in shape 
 and labeled with gold particles of 12 nm in diameter ( fig . 3b and 3d ) .
they showed lower electron - density than that of secretory 
 granules in enterochromaffine ( ec ) cells .
ec cells having polymorphous secretory granules 
 with the high electron density were observed in the intestinal epithelium , but gold 
 particles were not located on their secretory granules ( fig .
morphometrical analysis indicated that the shortest and longest 
 diameters of secretory granules of l - cells were 355  62 nm ( mean  sd ) and 287  48 nm , 
 respectively .
electron micrographs of l - cells in the chicken distal ileum . ( a ) low magnification 
 view of l - cell showing glp-1-immunoreactivity and located in villous epithelium . note 
 that its apical surface is covered with microvilli .
bar=1 m . ( b ) 
 high magnification view of a secretory granule from l - cell .
particles of colloidal 
 gold ( 12 nm in diameter ) are diffusely arranged on a granule .
( c ) low magnification view of l - cells showing glp-1 and 
 -2-immunoreactivity and located in epithelium of crypt .
bar=1 m . ( d ) 
 high magnification view of a secretory granule from l - cell .
both sizes of particles of 
 colloidal gold ( 6 and 12 nm in diameter ) are diffusely arranged on a granule .
( e ) high 
 magnification view of secretory granules of ec cell in chicken small intestine .
double immunocytochemistry showed that secretory granules of l - cells labeled with gold 
 particles of 12 nm in diameter were also labeled with those of 6 nm in diameter , showing 
 glp-2 immunoreactivity .
these two sizes of gold particles were diffusely arranged on 
 secretory granules ( fig .
the ratio between these two sizes of gold particles was 
 almost equal on a secretory granule of l - cells .
the present study clarified the colocalization of glp-1 with glp-2 as well as 
 ultrastructural features of l - cells in the chicken small intestine by colloidal 
 gold - labeling immunocytochemistry .
l - cells in the chicken small intestine are characterized 
 by microvilli covering their apical surface and many secretory granules showing moderate 
 electron density .
secretory granules without halo are relatively large and round or oval in 
 shape , and glp-1 and glp-2 are stored in them .
our previous studies at the light microscopic 
 level have reported the morphological features and the distributional pattern of 
 glp-1-immunoreactive cells in the chicken small intestine [ 11 , 12 ] . according to these studies , 

glp-1-immunoreactive cells show comma - like or flask - like shape and are in contact with the 
 intestinal lumen by their apical cytoplasmic process . in the present study , at the electron 
 microscopic level
, we also revealed that l - cells come into contact with the intestinal lumen 
 by microvilli covering their apical surface .
these findings indicate that l - cells are namely 
 open - type of endocrine cells and receive chemical signals in the intestinal lumen , such as 
 digests of ingested meal .
in fact , glp-1 is released in response to food intake and exerts 
 its physiological actions in mammals [ 2 , 7 ] .
our finding suggests that glp-1 is the meal - induced 
 hormone also in the chicken .
enteroendocrine cells have been classified into several types according to morphological 
 features , e.g. shape , size and electron density of their secretory granules .
some ultrastructural studies in the mammalian 
 intestine have shown that l - cells are characterized by round , homogenous , large ( about 300 
 nm in diameter ) and electron - dense granules [ 3 , 10 , 14 , 23 ] . in the present study ,
l - cells in the chicken 
 intestine were also characterized with secretory granules showing similar features to those 
 in mammalian l - cells mentioned above . electron density of secretory granules in chicken 
 l - cells , however , is not similar to those in mammals .
we identified ec cells together with 
 l - cells in this study and found that these ec cells contained polymorphous and electron 
 dense granules as reported in the previous study in the chicken duodenum . compared with secretory granules in ec cells , those 
 in l - cells
this difference in 
 electron density of secretory granules is likely to be one of characteristic features of 
 chicken intestinal l - cells .
human 
 glp-2 is a gastrointestinal hormone with 33% sequence homology to glucagon and shows the 
 intestinotrophic endocrine / paracrine actions . 

this peptide , consisting of 33-amino acids , is derived from proglucagon by its 
 posttranslational processing and secreted from intestinal l - cells .
thus , two forms of glp , 
 glp-1 and -2 , are synthesized from the same precursor and secreted from the same cells in 
 the mammalian intestine .
our immunocytochemical findings provide the prediction of 
 cosecretion of glp-1 with glp-2 from the same l - cells also in the chicken small intestine .
a 
 double immunofluorecent study demonstrated that l - cells showing the colocalization of glp-1 
 with glp-2 were mainly located in epithelium of crypts and lower part of villi of chicken 
 small intestine .
moreover , l - cells showing immunoreactivity for glp-1 only were observed in 
 epithelium of lower and middle parts of villi .
considering these findings at electron and 
 light microscopic levels , it is possible that glp-1 and -2 are costored in the same 
 secretory granules , but secreted from the same l - cells separately . in conclusion , l - cells in the chicken small intestine are characterized with microvilli 
 covering their apical surface and large secretory granules showing moderate electron 
 density .
colocalization of glp-1 with glp-2 in the same l - cells is also demonstrated , but it 
 is suggested that these two hormones are secreted from the same cells separately .
 abstractcolocalization of glucagon - like peptide ( glp)-1 with glp-2 in l - cells was investigated in 
 the chicken ileum by using double immunofluorescent and immunocytochemical techniques . 
 
 ultrastructural features of l - cells were also clarified in this study . 
 l - cells showing 
 immunoreactivity for both glp-1 and glp-2 were distributed in the whole ileum . 
 they showed 
 comma - like or flask - like shape and were located in epithelium of crypts and lower part of 
 intestinal villi . 
 l - cells showing glp-1-immunoreactivity only were found in epithelium of 
 lower and middle parts of intestinal villi . 
 transmission electron microscopy indicated 
 that l - cells identified by colloidal gold - labeled immunocytochemistry were covered 
 apically with microvilli , open - type and contained many secretory granules in their 
 perikarya . 
 these secretory granules without halo were round to oval in shape and showed 
 moderate electron density . 
 the longest and shortest diameters of secretory granules were 
 355  62 nm ( mean  sd ) and 287  48 nm , respectively . 
 double labeling immunocytochemistry 
 using two different sizes of particles ( 6 and 12 nm in diameter ) of colloidal gold 
 revealed that glp-1 colocalized with glp-2 in the same secretory granules . 
 this study 
 advances new morphological data about the endocrine system of the chicken small 
 intestine . 
MATERIALS AND METHODS
RESULTS
DISCUSSION