Functional Histology of Endocrine Cells in Bovine Intestine at Different Developmental Stages

Title

Functional Histology of Endocrine Cells in Bovine Intestine at

_{Different Developmental Stages( 本文（FULLTEXT) )}

Author(s)

PYAROKHIL, Asadullah Hamid

Report No.(Doctoral

Degree)

博士(獣医学) 甲第346号

Issue Date

2012-03-13

Type

博士論文

Version

publisher

URL

http://hdl.handle.net/20.500.12099/42930

※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。

Functional

Histology

_{of Endocrine}

Cells in Bovine

Intestine

at

Different

Developnental

Stages

(&#3BtR50

#Q)BB

tTLB

81

8

_{Fq3ji&haFn&Q)i&eEgAnSiB9#*}

)

-:''.

:I:.I;I

L _ .i 2011

(?

7u

5p

The Uhited Graduate School _{of Veterinary} Sciences, Gifu Uhiversity

(Obihiro

Uhiversity _{of Agriculture and} Veterinary

_Medicine)

Table

_of

Contents

Chapter 1

General introduction

1.1. Overview...

...1 1.2. Chemical Signals including Regulatory Peptides _{of the Gut...2}

1.3. GeneralMaterials _{and methods...6}

Chapter 2

Immunohistochemical _study on the _ontogenetic development _{of the regional}

distribution _{of peptide} W, pancreatic polypeptide, and glungon-like peptide I

endocrine cells in bovine gastrointestinal tract

2.1. Introduction...ll

2.2. Materials _{and methods...}

...12

2.3. Results..."...13

2.4. Discussion...-...18

Chapter 3

Quantitative

Immunohistochemical Study _{of Endocrine} Cell Distribution in the

Bovine Large Intestine

3.1.Introduction...

...38 3.2. Materials _{andmethods...39}

3.4. I)iscussion...41

Chapter 4 The developmental _{plasticity of colocalization pattern of peptide} W _and glucagon-like peptide- 1 in the endocrine cens of bovine rectum 4.1. Introduction... ...56 4.2. Materials _{andmethods...57} 4.3. Results..."...58 4.4. Discussion...59 Chapter 5 Generaldiscussion...65 Abstract... ...70 AbstractinJapanese...74 Acknowledgements.. References...-...80

Chapter

I

General

introduction

1. I. Overview

Endocrine _{cells distributed} in _{the gastrointestinal} tract _{produce various}

kinds _{of signal materials} to _regulate diverse functions _{ofgut such} as _secretion,

Secretion and motility

(65,78).

Many studies have elucidated the distribution

of various endocrine cells in the gastrointestinal tract of different mammals including domestic _{animals such} as _cat

(40),

_cattle

(39),

_sheep

(12),

horse

(38),

pig

(36),

water buffalo

(8, 52),

and camel

(16).

These studies have aimed to

demonstrate their distribution _{and relative} frequencies in different _{parts of}

the _{gastrointestinal} tract _and to _understand their functional _roles in the

digestive _system. Several _{peptides produced} by _{endocrine cells} were _newly

identified in _recent decades. _{In addition,} Some kinds _{ofgut peptides} including

PW _and GLP-1 were distinguished to _participate in the control mechanism

within the gut as _wen as _outside it such aS _{nutritional and} food intake

regulation

(30)

which has

high

significance in the domestic animal husbandry. The _ruminant has drastic _changes twice _{in nutrition,} from fetus to

postnatal, and suckling to herbivorous. IIowever, no _study has been focusing

relative frequency of endocrine cens in the gastrointestinal tract of cattle from _early fetus to _adult cow. Furthermore, it is intended to _{emphasize the}

regulatory role of the large intestine because ruminants are _{usually noticed}

on _{the stomach.}

The _{present study} investigated the distribution _{of endocrine cells} in the

gastrointestinal tract of cattle at pre- and post-natal stages with different developmental _stages

_{(Chapter 2).}

It was focusing on the large intestine by

the _precise investigations on the detailed _{segments of the convoluted gut}

(Chapter 3).

Furthermore, the _{detail study} was done on the

developmental

plasticity of the colocahzation pattern of gut hormones

(Chapter 4).

The

present study would play a key _role to _understand the control mechanism _of

the bovine digestive tract from the view _{points of the developmental plasticity}

of the regulatory systems.

1.2. Chemical Signals including Reguhtory Peptides _{of the Gut}

In this section, brief descriptions are _made on the _{characteristics of}

chemical signal materials which are dealt in the _{present study.}

Peptide W

(PYY)

is a _{member of PP} family _along with _pancreatic

polypeptide

(PP)

and neuropeptide Y

(WY)

(24).

These three peptides share

similar chemical structure based on the number _{of amino acids}

(36-amino-acid residue

_peptides).

The regional distribution of PW has been studied in a

radioimmunoassy of tissue extracts. Lundberg et a1.

(53)

first localized PYY

to a _{population of endocrine cells} in the intestinal mucosa _of a _{variety of}

species, including man. PW immunoreactive _cells are _present in the distal

small intestine, colon and rectum, but were rare _{and absent} in _{the stomach}

and duodenum. Immunohistochemical and tissue extraction studies

subsequently confirmed the distal pattern of distribution of PW in a _number

of species

(I,

21, 22, 23,

81).

Recent studies have demonstrated that PW1.36

and PW3.36 are the

major

molecular forms of the peptide in tissue and in the

circulation

(15).

PYY is present in

high

concentration in mucosal extracts of the human ileum, _{colon and} rectum

(1).

PW is a _{multifunctional} hormone;

the _main function is to inhibit _{gastric emptying and mediates the effects of}

deal fat on _{gastric motility} as indicated in _rodents. PW has been identified

as a _peptide involved in the ileal brake

(50,64).

The temporal _{pattern ofPYY}

release is different from that of other gastrointestinal peptides with

suggested roles in feeding control. Plasma levels of PW gradually increase

aaer meal initiation, reaching a _{peak at about} 60 _{minutes, and}

high

levels

are _maintained for a _{number of hours after} a _meal

(61).

PP is a _{structurally related member of the} PP family. PP isa 36 amino

acid peptide predominantly expressed in the endocrine pancreas. However,

rare PP-immunoreactive _{endocrine cells have} been described in some but _not

PP _celk are _{also present} in the intestinal mucosa, _especially in the ileum _and

colon

(10,

47,

49).

PP secretion is stimulated by food ingestion and exercise,

and vagal tone is an important determinant _regulating PP _secretion in

rodents and human

_{subjects (14).}

The main function of PP is to inhibit

pancreatic exocrine secretion and relax the gallbladder

(54).

PP is considered

as food intake _regulator

(14)

_{and also plays} an important _{role in negative} feedback _{control of the pancreas,} inhibiting _{netually mediated stimulation of}

this

gland (54).

Glucagon-like _peptide- I

_{(GLP- I)}

belongs to a large family _{of glucagon}

(34, 37),

which is secreted from L-cells of the gastrointestinal mucosa

(34,

37,

79).

GLP-1-producing L-cells have been identified in the

jejunum,

neum _and

colon, with the

highest

cell densities reported in the ileum and colon

(34,37,).

GLP- 1 is _{secreted after nutrient} ingestion _{and stimulates} insulin secretion in

a _glucose dependent manner

(42,

56,

63).

GLP-I functions as a _gut hormone that _{helps regulate} blood _{glucose and} feeding behavior _{in mammals.} GLP-I is

involved in the ileal brake _mechanism. Gastric inhibitory _and intestinal

motility actions of ileal nutrients are _{partiany mediated}

through

GLP-1

release. On the other hand, GLP-1 increase insulin release and reduce

appetite in human

(30).

GLP-1 is considered to be a

biologicany

_and therapeutically interesting hormone, because _of its _potential

Chromogranin

(CG)

is an _{acidic protein widely} distributed in

entero-endocrine cells and in other members of the paraneuron family. Therefore,

CG has been _claimed as a common "marker" _{of an neuro-endocrine cens}

(13,

25,

_28).

As to the _{gastro-entero-pancreatic}

(GEP)

_endocrine system, previous

studies localized CG in a _{variety ofendocrine cells}

(ll,

13, 17, 25, 66, 85,

90).

Evidence increases that CG in this location _may serve as a _precursor for

other hormones to be identified

(18,

19, 35, 41, 71, 70, 73,

81).

The main

physiological

function of CG is not yet clearly understood, but studies

suggested that CG and other related proteins

_might

function in the

organization of the granule matrix

(e.g.

binding of

_calcium),

in the processing

or

_packaging

_{of peptide} hormones or their _{precursors, and possibly} in the

regulatory mechanisms after secretion

(41,

72, 9

1).

Serotonin

(Ser)

is a _{regulatory amine of mucosal enterochromaffin}

(EC)

cells. EC cells constitute the largest endocrine cell population in the

gastrointestinal tract and produce over 90% _{of an the Ser synthesized} in the body

(4).

_{Ser plays} an important _role in the control of gastrointestinal _smooth

muscle contraction and epithelial secretion

(26).

Somatostatin

(Son)

is a _peptide hormone _{symihesized and secreted} by

endocrine D cells. Son cells are _widely distributed in the _{gastrointestinal}

mucosa, _pancreatic islets and in the autonomic _{and central} nervous _system

biological _activities in different _{parts of the} body _such as the _{secretion of}

pituitary hormones, release of a _{wide variety of peptides} hormones from _all

regions of the gut and endocrine pancreas, gastric acid Secretion, pancreatic

exocrine secretion, intestinal absorption, proliferation and gastrointestinal

motility

(51, 57).

1.3. General Materials _{and methods}

The _{present experiments} on _animals were _{carried out} in _accordance

with the guideLnes of the Committee of Animal Exp.eriments of Obihiro University _{of Agriculture and} Veterinary Medicine

(No.

17-51, 20-95, 21-lO8,

23-46).

Totany

_forty-eight

Holstein _cattle in different _{pre- and postnatal}

stages were _used in this _study. Prenatal _animals were _{obtained at} the

autopsy of their euthanized mothers for pathological inspection. The prenatal

animals

(fetuses)

were divided into 3 developmental stages according to their

crown-rump length

(CRL):

early fetus

(CRL:

20 - 40

cm),

mid fetus

(CRL:

41

-70

_cm),

_and late fetus

(CRL:

71

-100

_cm).

The _{postnatal animals} were divided

into four developmenta1 _{Stages according} to their _{age: suckling calf}

(1

-

2-week-old and

5-7-week-old),

2. weaning calf

(2-month-old),

3. weaned calf

(7-month-old and

10-month-old),

and 4. adult cattle

(1

-8-year-old)(Table

1.

_1).

The _{postnatal animals} were _{exsanguinated} from the carotid _{artery under} the

anesthesia

(xylazine

hydrochloride 0.3mgnig and thiopenta1

7mgnig)

and dissected.

Tissue _samples were taken from the _{whole gastrointestinal} tract;

esophagus

(upper

and lower

_part),

rumen,

_r6ticulum,

omasum, _oxyntic

(body)

and pyloric parts of abomasum

(glandular stomach),

duodenum

(cranialpart),

jejunum

_{(mid part),}

ileum

_{(terminal part),}

cecum

(body),

_{ascending colon}

(proximal

_{loop, centripetal} turns, _central nexure, _centrifugal turns, dista1

loop),

transverse _{colon, descending colon, sigmoid colon}

(cranial

to _{the rectal}

ampulla)

and rectum

(ampulla

and

just

cranial to the anorectal

lme).

The

samples were then fixed in _the Bouin's _{solution at} room temperature

(RT)

for

24 hours

(h).

The _{obtained samples} were trimmed into _{small pieces and} were

dehydrated in _{ascending concentration of ethano1, cleared} by _{xylene and}

embedded in parafBn

(Paraplast

Plus@, Kendall, MA,

USA).

The samples

were then cut at 1-4pm thickness used sliding microtome

(Leica

SM 2000 R,

Germany, _with blades S35 Feather,

_Japan),

_collected on _{gelatin coated slides}

and kept

_overnight

in the warming cupboard

(40oC).

Hematoxylin _{and eosin}

(HE)

staining was _performed to investigate the

general histological inspection of the gastrointestinal tract.

Immunohistochemical _staining was _applied for the detection _{of targeted}

endocrine cells with specific antibodies. The sections were deparaffinized _and

rehydrated. The endogenous peroxidase activity was blocked _with 0.3% H202 in methanol, and washed three times by phosphate buffer saline

(PBS,

pH

normal goat serum

(I:50,

S-1000, Vector Laboratories Inc., Burlingame, CA,

USA)

f.r 30 _{min at} RT. The _secti.ns were _{again washed} by PBS yand

incubated _with targeted primary antisera and kept

_overnight

in refrigerator

(approximately

5

_oC).

The dilution tests _{of prlmary antiserum} were

performed to get the optimal dilution fTor the best condition of specific immunoreactivity before _applying. In _{the second} day, the _sections were

washed by PBS and incubated with secondary anti-rabbit IgG raised in goat

(1:200,

BA-1000, Vector Laboratories

_Inc.)

for 30 _{min at} RT. Aaer _washing

again three times by PBS, the sections were further incubated _with the

avidin-biotin peroxidasae complex

hBC)

method using Vectastain EEte ABC

kit

(Vector

Laboratories,

_Inc.).

Immunoreaction _sites were _revealed by Tris

buffer

(pH7.4)

containing 0.02% 3, 3'-diaminobenzidine tetrahydrochloride

and 0.06% H202. The Sections were

lightly

_{counterstained} with hematoxy1in

and dehydrated in ascending concentration of ethanol, cleared by xylene and then _mounted.

Each _ofimmunoreative _cellsin different _{gastrointestinal portions} were

observed under the conventional

light

microscope and photomicrographs

were taken by a digital camera

(DS-5M,

Nikon, Tokyo,

Japan).

For the cell

counting, special counting chamber with the counting grid was &ed in the

eyepiece of the microscope and the number of immunoreactive cells was

estimated in ten random unit areas _{per section of each gastrointestinal part.}

only with clear and identifiable nucleus were _{counted and recorded} for further _analyzing.

The _obtained data were _{arranged and calculated} by Microsoa Excel

2010, _and the _values were _{statistically analyzed} by GraphPad Prism

(version

5.00, GraphPad SoRware, San Diego, Cahfornia,

_USA)

_using

one-way ANOVA followed by Tukey's post hoe multiple comparison tests. The

values were _expressed as Mean i SEM _and differences were _considered

Table 1. I.The detail information _{of cattle used} in the present study

Develop _mental stage Number of animals

CRL/age Chapter _{2 and 4} Chapter 3

Early Betus Mid fetus Late fetus Suckling _calf Weaning _calf Weaned _calf 20-40cm 41-70cm 71 -lOOcm 1.2weeks 5-7 weeks 2 months 7 _months 10 months I -8years 9 8 5 5 CRL: Cmwn-rump length

Chapter

2

Immunohistochemical

_study

on

the

_ontogenetic

development

_of

the

_regional

distribution

_{of peptide}

W,

_pancreatic

_polypeptide,

and

glucagon-like

peptide-

I

endocrine

cen8

in bovine

gastrointestinal

tract

2. 1. Introduction

Endocrine _{cells dispersed} in the _{gastrointestinal} tract _{comprise the}

largest _{endocrine organ of the body.} It is _{composed of}more than 20 different

cell populations

(78).

These gastrointestinal endocrine cells synthesize and

release various types of gastrointestinal hormones to regulate the digestive

system and the body

(78).

In recent decades, peptide W

(PW)

and

glucagon-like peptide-1

(GLP- 1)

have been _proven to influence feeding _mechanisms in

rodents and human

(24,

77,

93).

PW is a _{member of the pancreatic polypeptide}

&P)

family

(83),

_which

includes _neuropeptide Y

(NPY)

_and PP, all _{consisting of 36 amino acids.}

NPY is found _{only within} the brain _{and peripheral} nervous _{system, while}

PW _and PP are found _{mostly within endocrine cens of the}

gastro-entero-pancreatic system. PW is Synthesized and released from the endocrine cells

populations of cells located at the periphery of pancreatic islets

(58,76).

PP

cens are _aho distributed _at a low level in the exocrine pancreas and in the

gastrointestinal tract, mainly in the colon and rectum

(39,

78,

83).

GLP-1 is one _{of the members} that are _produced from _proglucagon

sequence

(37)

Large numbers of GLP-1 endocrine cells have been identified in

jejunum,

ileum, _{and colon}

(77,93).

It has been _reported that PW _and GLP-1

have important _roles in _{the regulation of appetite and} feeding, _and have an

additive effect on feeding _control in _{rodents and} human

(59,

77,

93).

However,

the

_{physiological}

importance _{of these} three _{peptides in ruminants} is yet to be

determined

_{(61, 62).}

There have been no _studies on the _ontogenetic

development _{of these} three kinds _{of endocrine celk} in domestic _animals. The

present study was _conducted to _reveal the

_regional

distribution _{and relative}

frequencies _{of PW,} PP, _and GLP-1 _{endocrine ceb} in the gastrointestinal

tract _{of pre- and postnatal cattle.}

2.2. Materials _{and methods}

In this _study, twenty-one Hohtein _cattle in the fonowing seven

ontogenetic stages were _examined. The detail _{of animals and method of}

sampling were described in the section of general _materials in Chapter I. Brief _explanations are fonows: _early fetus: 20-40 cm in _crown-rump length

(CRL, n=3),

mid-fetus: CRL 41-70 cm

(n=3),

late fetus: CRL 71-loo cm

(n=3),

calf

(10-month-old, n=3),

and adult

(I-8-year-old,n=3).

Tissue samples were obtained from esophagus, rumen, _reticulum, omasum, _abomasum, duodenum

(cranialpart), jejunum (mid-portion),

ileum

_{(terminal portion),}

cecum

(body),

colon

(central flexure),

and rectum

(terminal

portion,

just

cranial to the

anorecta1

line).

The _{detail method of paraffin} section and immunohistochemistry were

explained in the Chapter 1. The primary antisera used in this chapter were

raised in rabbit against porcine PW

(I:10,000,

IHC7173, Peninsula Lab. Inc., Belmont,

_USA),

human PP

(1:10,000,

YO80, Yanaihara Institute, Inc.,

Shizuoka,

_Japan)

_and human GLP-1

(1:10,000,

Y-320, Yanaihara

_Institute).

2.3. Results

The _{present study} demonstrated _{the characteristic} distribution _ofPW-,

PP-, _and GLP- 1-immunoreactive _{endocrine cells, whereas} immunoreactivity

for these peptides in the nervous _system was _{not revealed.} Immunoreactive

endocrine cells were _not found in the esophagus, rumen, _reticulum, omasum,

or _{abomasum of all} seven _{ontogenetic groups.} They were detected in the

sman and large intestines of all groups at different frequencies. The

mentioned immunoreactive cells were _mostly detected in the basal

_region

_of the intestinal _{crypt glands and} a few numbers were _{also detected} in the vinus

with the presence of cytoplasmic processes ending with the lumen. Round and

spherical shaped cells were _also found _rarely in different

_regions.

The frequencies _of PW-, PP- _and GLP-1-immunoreactive _cells were _vary

depending on intestinal

_regions

_{and perhaps} food habits _{of animals} at

different developmental _ages.

Dispersed PW-immunoreactive _cells were detected _mostly in the large

intestine, _especiany in the _rectum, but _only a few _cens were detected in the

small intestine at allstages. PW-immunoreactive cells were detected in the basal

_regions

_{of intestinal crypt glands and} a few numbers were _{also detected}

in the _{villus glands.} PYY-immunoreactive _cens were _{mostly spindle shape}

with long cytoplaSmic processes reached the intestinal 1umen. Few cens with the oval, round and spherical shapes were _{also found} in different _regions.

PYY-lmmunoreactive _celh were immunohistochemicany detected

abundantly in the prenatal

(early,

mid and

_early)

stages. In early fetal stage,

PW-immunoreactive _cenS were _not detected in the duodenum _{portion of the}

small intestine. They were detected from

jejunum

to rectum parts. The most

abundant immunoreactivities were _observed in the ileum

(Fig. 2.1A)

_and

rectum

(Fig.

2.

1B)

parts. In mid fetal stage, PW-immunoreactive cells were

distributed _{almost similar} to that _{of early} stage, but their frequencies were

higher

in mid fetal stage. Very few PW-immunoreactive cells were detected

in the

jejunum

of mid fetal stage. PW-immunoreactive cens were _observed

(Fig. 2.2C)

and rectum

(Fig. 2.2D)

parts of the mid feta1 Stage.

PYY-lmmunoreactive _cells were _also detected in the late fetal _stage; their

frequencies in _{the late iTetalstage} were _{also similar} to _{that of early and}

mid-fetal _stages. However, in the late fetal stage, PYY-immunoreactive cens were

also detected with a _very low frequency in the duodenum _portion.

The frequency _and distribution _{of PW-immunoreactive cens} were low

in the _postnatal

(sucklmg,

weaning, weaned and

_adult)

stages.

PYY-immunoreactive _cells were _not detected in the duodenum _{of suckling,}

weaning, weaned and adult stages. Very rare PW-immunoreactive _cens were

observed in the

jejunum

of suckling, weaning and weaned stages. However,

PW-immunoreactive _cells were detected from the ileum to the _rectum

portions of all postnatal

(suckling,

weaning, weaned and

_adult)

stages. In

suckling stage, the distribution of PW-immunoreactive cens was

higher

in the ileum

_{(Fig. 2.3A)}

_and rectum

(Fig. 2.3B)

portions. The distribution of

PW-immunoreactive _cens was _almost same in the

jejunum,

ileum, cecum

and rectum of weaning, weaned and adult stages. However,

high

number of

PW-immunoreactive _cells was _observed in the rectum portions of weaned

(Fig.2.3C)

and adult

(Fig.2.3D)

stages.

The

_regional

distribution _of PW-immunoreactive _celh was

significantly

(P

<

_{0.05) higher}

in the

rectum at mid fetus

(16.27

i

1.7l),

differences _among the intestinal segments were _observed in _{early and} late

fetusland _{weaning calf stages}

(Table

2.1, Fig.

_2.10-ll).

With _regard to _{ontogenetic stage,} on the other hand, the frequency _of

pw-immunoreactive _cells was _{significantly}

(P

<

_{0.05) higher}

_{in the ileum}

of the early fetus

(7.60

i

_0.25)

_and the _{rectum of the mid} fetus

(16.27

i

_1.7l)

than _{at other} stages, while in other

_regions

of the gut, such a _marked

tendency was not observed

(Table

2-1, Fig. 2.

12-13).

Open-type PP-immunoreactive _cens were detected in the intestinal

crypt

gland

with the spindle, oval and spherical shapes. In the small

intestine, PP-immunoreactive _cells were _not detected in the duodenum part.

However, _very few PP-immunoreactive _cens were detected in the

jejunum

_of

prenatal

(early,

mid and late

feta1)

and postnatal

(sucklmg

calf and weaned

calf)

stages. pp-immunoreactive cells were _not detected in the

jejunum

_of

weaning calf and adult stages. PP-immunoreactive cell were _mostly detected from the ileum to the rectum portions of allpre- and postnatal stages.

In _{the prenatal stages, the distribution of} PP-immunoreactive _cens was

higher

in _{the mid and late fetal stages.} However, in _{the postnatal stages,} they

were detected with _very low number from the ileum to the rectum portions.

In _{the suckling stage,} PP-immunoreactive _cells were _{clearly observed} in the

ileum, _{colon and rectum portions}

(Fig. 2.4A-C).

PP-immunoreactive _{cells, in}

the _{weaned and adult} stages were _{mostly observed} in the colon and rectum

PP-immunoreactive _cens were _observed between _any intestinal _{segments at}

different developmental _stages

(Table

2. 1, Fig. 2.10-

_13).

Relatively _abundant GLP- 1-immunoreactive _cens were _mostly detected

in the crypt

glands

of small and large intestines at all developmental stages.

GLP-I-immunoreactive _cells were _{all open-type with} the _{Spherical and}

spindle shapes, which had long cytoplasmic processes ended with the lumen

similar to PYY-immunoreactive cells.

The localization _{of GLP-1-immunoreactive cens} was

higher

in the

prenatal

(early,

mid and late

fetus)

stages than in that of postnatal

(suckling,

weaning, weaned and

_adult)

stages. GLP-1-lmmunoreactive ceh, in the early

and mid-fetal stages, were more numerous in the duodenum

(Fig. 2.6A-B)

and rectum

(Fig.2.6C)

portions. GLP- I-immunoreactive cells in the late fetal

stage were detected in all _portions. However, they were more numerous in

the small intestine

(Fig.2.7A-B).

GLP-I-immunoreactive _cells were _{also moderately} detected in _all

portions of the postnatal

(sucklmg,

weaning, weaned and

_adult)

stages. Their

immunohistochemical distribution was _{almost slmilar} in the _{suckling and}

weaning stages. However, GLP- 1-lmmunoreactive cens were _observed

higher

in the duodenum _{of the suckling calf stage}

_(Fig.2.8A).

In _{the weaned stage,}

GLP-I-immunoreactive _cens were

higher

in the small intestine

(Fig.2.8B-D).

The

_regional

distribution _of GLP-1-immunoreactive _cells was

significantly

(P

<

0.05)

increased in the duodenum

(6.00

i

_0.70)

_{and rectum}

(5.66

i

_I.25)

_{of sucklmg calf,rectum}

(5.56

i

_2.01)

_{of weaning calf, duodenum}

(3.80

i

_0.96),

_ileum

(3.63

i

_0.53),

and rectum

(3.77

i:

_0.50)

_{of weaned calf, and}

rectum

(5.63

i

1.67)

of adult compared with those of other intestinal

_regions

(Table

2.1, Fig. 2.

_10-ll).

The distribution _{of GLP-} 1-immunoreactive _cens was

also significantly

(P<0.05)

increased ontogenetically at mid fetus stage

(13.33

i

_1.77)

in the duodenum, _{mid and} late fetus _stages

(8.26

i 1.33, 9.06 i

2.03)

in the

_jejunum,

_mid fetus _stage

(ll.6

i

_2.03)

in the neum, late fetus _stage

(7.73

i

_2.36)

in the cecum, _{mid and} late fetus _stages

(9.46

i 1.75, 8.93 i

_2.46)

in _{the colon, and mid} fetus _stage

(15.73

i

_I.54)

in the rectum

(Table

2.1, Fig.

2.12-13).

2.4. Discussion

In the present study, the

_regional

distributions of PW-, PP-, and

GLP-1-immunoreactive _cells were for the first time investigated in the

gastrointeStinal tract of cattle at different ontogenetic Stages. The present

results indicate that PW, PP, and GLP-I endocrine cells were _absent in the

esophagus and stomach. However, they were _widely distributed in _{the small}

and large intestines of all cattle with different relative frequencies depending

Different _{endocrine cells have} been _studied in _{the gastrointestinal} tract

of various mammals including human

(74),

cat

(40),

cattle

(39),

sheep

(12),

horse

(38),

pig

(36),

water buffalo

(8,52)

and camel

(16)

to demonstrate their

distribution _{and relative} frequencies in different _{parts of the gastrointestinal}

tract _and to _understand their functional _roles in the digestive _system.

However, no detail studies on distribution _{of PW,} PP _and GLP-1 _endocrine

cells in different pre and postnatal stages of neither human and rodents nor

ruminants we _{reported yet.} Thus, the present study could be a key _paper for

the distributions _{of mentioned endocrine cens} in _{the gastrointestinal} tract _of

cattle. The distribution and frequencies of PW, PP and GLP-1 endocrine

cells will be individuany discussed with the available previous studies on

ruminant species.

In the present study, PYY-immunoreactive cens were detected in _an

portions of the intestine with different regional frequencies; they were more

numerous in the large intestine. Previous _studies demonstrated

PW-immunoreactive _{cens mostly} in the distal _{portions of the small} intestine _and

in the large intestine

(2,

3, 8, 52,

_62).

_{In cattle, PW-immunoreactive ceus}

were _much less abundant in the ileum than in the large intestine, _such as in

colon and rectum, aS has been _reported in the intestine _{of babiruSa}

(2),

_water buffalo

_{(8, 52)}

_{and sheep}

(62).

PW-immunoreactive _cells were _not detected

(philippine

_water

_{buffalo) (8),}

_while they were numerous in the large

intestine, _especially in the rectum of carabao

(8).

The _{role of} PYY _peptide in the _{gastrointestinal} tract has been widely

investigated in _{monogastric species, especially} in human

(7).

The _nutrient

infusion into the ileum inhibits

_jejunal

_motnity

_{(75, 87)}

_and decreases _antral

and duodenal peristaltic pressure wave

(27),

the _so-called "ileal brake". Moreover, it has been _reported that _colonic food infusion _reduced ileal

motility

(88)

and pancreatic secretion

(32)

with increasing PYY and GLP-1 in the colon

(88).

In rats, cecal nutrient infusion reduced food intake more than

ileal infusion

(55),

_suggesting the importance _{of the} large intestine for the

regulation of food intake. Onaga et a1.

(62)

reported controversial findings for the ileal brake in _sheep. At the same time, however, they _reported the

increasing _{abundance of PW content} in _{the dista1 large intestine. In cattle,} it

is _assumed that _abundant PW-immunoreactive _cells in the distal _{portions of}

Small intestine and in the large intestine

_might

be involved, at least in part,

in the dista1-to-proximal intestinal feedback. However, in _ruminants, foregut

fermenters, _{actual regulatory mechanisms} are _stillunclear

(6

1,

62).

In the bovine _{ontogenetic results,} PW-immunoreactive _cens were

decreased in _{postnatal stages.} This decreasing tendency in the _postnatal

period is consistent with results of a _{previous study} on the _{ontogeny of gut}

endocrine cens in water buffalo

(52).

They found that the frequency _of

buffalo than in the 5-month _and 5-yearold buffalo. It is _{suggested that other}

intestinal _{epithelial cens such} as _{goblet and absorptive epithelial cens show} a

greater increase in _{number with} intestinal development _after birth. The

decreasing tendency _{of PW-immunoreactive ceus} was _{prominent at} the

weaning period as _wen as _nursing. The _{adaptation of digestive system} to the herbivorous _{nature may also have} drastic influence _{in the regulatory system}

of the gut.

At all developmental stages of the cattle, PP-immunoreactive cells were

sparsely distributed in different intestinal portions. Similar to PYY-immunoreactive _cen distribution, PP-immunoreactive _{cells also tended} to be

increased in the large intestine. However, no _significant differences were

observed among any intestinal regions of the seven _{stages examined} in the

present study. Similar to the present study, small numbers of

PP-immunoreactive _{cells in} the distal _small intestine _{and relatively} large

numbers in the large intestine were _reported in _{calf and} cow

(39),

as _well as

sheep

(12).

However, intestinal PP-lmmunoreactive cens seem to differ depending on the _{anlmal species.} In human _{colon and rectum,} few

PP-immunoreactive _cells were detected

(74).

Moreover, _rat PP-immunoreactive

cells were transiently _expressed for a _short time in _{the endocrine cens of}

colon at the postnatal stage

(20).

It is thus _assumed that the functional _roles

to _reduce food intake _{and may} induce long term _{suppression of appetite}

_(74).

However, the _{main role of} PP _{in ruminants} including _{cattle remains unclear.}

In the present study, numerous GLP-I-immunoreactive _cells were

detected in _{all parts of the small and}

_large

intestines _{at all developmental}

stages. GLP-1-immunoreactive cells were more numerous _at the prenatal

stages than at calf and adult stages. The relative frequencies of

GLP-I-immunoreactive _{cells tended} to be

_higher

in the duodenum _{and rectum of the}

intestine. The _general tendency _{of the distribution of GLP-} 1-lmmunoreactive

cells in the large intestine was basicany _Similar to those _of

ghcentin-lmmunoreactive _cells in _{calf and} cow

(39),

as _well as _sheep

(12).

This _may be

related to the fact that GLP-1 and glicentin are derived from the same

precursor molecule

(37).

GLP- 1 is _{secreted in the distal small intestine and colon by stimulation}

of carbohydrate and fat, and inhibits gastroduodenal motility

(69)

and gastric

acid secretion

(89).

Therefore, it is considered that GLP-1 also has an

important _role in ileal brake _and/or dista1-to-proxlmal feedback, like PYY,

and the two _{peptides may act cooperatively}

_{(59, 89).}

The reasons for the decreased frequency _{of GLP-1-immunoreactive cells with} development _may

be the same aS those for PW-immunoreactive _cells.

The _{present study} demonstrated _{the regional distribution and relative}

frequencies _{of PW-,} PP-, _and GLP- I-immunoreactive _cens in the intestine _of

frequency _{of these endocrine cells vary among} different intestinal _segments

and at different developmental stages in cattle. These differences of PYY-, PP-, _and GLP-1-immunoreactive _{cell distribution}

might

be due to the

stage-dependent _changes in intestinal _{growth, secretion, motility, and} diet. Further

studies on the

_{physiological}

functions _{of these} three kinds _{of hormones} - PW,

PP, _and GLP-I - in

ruminants are _needed. The _{present results} on the

ontogenetic distribution of three types of endocrine cells in the

gastrointestinal tract of cattle should provide the basis for future extensive

∼.

-

'L-I

∼__i&_

∼

Fig. 2.I. Peptide YY-immunoreactive _cells in the ileum

W

_{of early fetus}

(CRL

26

Jl i:. =itL3)..I:..7

y-'u--- ----Wti3r I.gi8B3ZF1/A

Fig. 2.2. Peptide W-inmunoreactive _cens in _the ileum

W,

cecum

(B),

_colon

(c)

_{and rectum}

(D)

_{of mid} fetus

(CRL

55

Fig. 2.3. Peptide W-immunoreactive _ceus in _the intestine _{of cattle, A: ileum}

of suckling calf

(12-day-old),

B: rectum ofSuck1ing calf

(12-day-old),

C= rectum

_i!Ei

-_

ij@;iL

∼T

Fig. 2.4. Pancreatic _{polypeptide-immunoreactive cells} in the intestine _of

cattle. A: ileum of suckhg calf

(12-day-old),

B: rectum of Suckling calf

rib Tll i I

.I.S::

i i

Fig. 2,5. Pancreatic _{polypeptide-immunoreactive cells in} the intestine _of

cattle. A: colon of cow, B: rectum _ofcow, C: _{rectum of weaned calf}

Fig. 2.6. Glucagon-like _{peptide-1-immunoreactive cells} in the intestine _Of

cattle. A: duodenum of mid fetus

(CRL

55

_cm),

B: duodenum of early fetus

- -' _I I. _: I iA I I I l I I I Rttt. LL'_ t'.

'Tt5

a

,:k

Fig. 2.7. Glucagon-Eke _{peptide-1-lmmunoreactive} endocrine cells in the

intestine _{of cattle. A: duodenum of late fetus}

(CRL

96

_cm),

B:

jejunum

of late

fetus

(CRL

90

Fig. 2.8. Glucagon-like _{peptide-I-immunoreactive cells} in _the intestine _Of

cattle. A: duodenum of suckling calf

(12-day-old),

B: duodenum of weaned calf

(10-month-old),

C=

_jejunum

_{of weaned calf}

(10-month-old),

D= ileum _{of weaned}

I?r- :Tif7PL-&.7E::.T_{;:a-TTTEVk7f7'W.1:.;.qT_5F}

ty

.yT.:.{ ( iI"Ill....A. .

I

Fig. 2.9. Glucagon-like _{peptide-I-immunoreactive cells} in the intestine _of

DtIO Jej ne Ccc Col Rcc (c) 1S 16 ∼ 14

S12

l!

":

5:

2 0

Duo _Jcj ne eec Col Rcc htestine

t= PYY = PP II GLP-1

Fig. 2.10. Distribution _{and relative} frequencies _{of endocrine cells per unit}

area

(625 Llm2)

in the intestine _{of prenatal}

A:

_early fetus, B: mid fetus, and

c: late

_fetus).

PW: _peptide W, _{PP: pancreatic polypeptide,} GIJP-1:

glucagon-hke _peptide-1. Duo: duodenum,

Jej: jejunum,

Iie: ileum, Cec: cecum, Col:

a I,I cl

I-g

U cI L< cq 'a 5 U

Duo _Jej []c eec Cot Rec 18 16 14 12 10 l8 16 14 12 10 8 CaJ f_{(w eancd)}

Duo _Jej Ile eec Col Rec

(a) 18 16 cl l4 4J 512 ･-a10

f

∼ 5 6 U ₄ (D) l8 16 d14 V 512

I-i

I:

% 6 U 4

Cat f_{(yveani ng)}

Duo _Jej ne eec Col Ref

DtLO _Jej ne eec Col Rec htesti ne

t= Pry a PP )GLP-1

Fig. 2.ll. Distribution _{and relative} frequencies of endocrine cells per unit

area

(625 pm2)

in the intestine _{of postnatal}

h=

_suckling calf,B: weaning calf,

c: weaned calf, D:

adult).

PYY: peptide YY, PP: pancreatic polypeptide,

GIJP-1: _{glucagon-like peptide-1.} I)uo: duodenum,

_{Jej: jejunum,}

Ile: ileum, Cec:

･B,

::

a::

31:

5

6 4 2 0 18 (C) l6

{L

I.:.

.i.I:

5

6 4 2 0 4 5 6 7 Developmental _stiLgeS D PYY D PP - GLP-1

Fig. 2.12. Distribution _{and relative} frequencies _{of endocrine cells} per unit

area

(625 pm2)

in the _small intestine

A:

duodenum, B:

Jejunum,

and C:

ileum)

of seven developmental _{Stages of cattle}

(l:

_{early fetus,} 2: _mid fetus, 3:

late fetus, 4: suckling calf,5: weaning calf, 6: weaned calf and 7=

_adult).

PW:

18 (J)) l6

i

:I:I

'U:

a

6 4 2 0 4 5 6 7 Develop-ental stages E= PYY Q PP Ill GLP-I

Fig. 2.13. Distribution _{and relative} frequencies of endocrine cells per unit

area

(625 pm2)

in the large intestine

h=

cecum, B: colon, and C:

_rectum)

_of

seven developmental _{Stages Of cattle}

(1:

_{early fetus,} 2: mid fetus, 3: late fetus,

4: _suckling

_calf;

5: _{weaning calf, 6= weaned calf and} 7=

_adult).

PW: _peptide

Table 2. 1.Distribution _{and relative frequency ofPW-, PP-, and GLP-} 1-immunoreactive _cells

in the bovine intestine at different developmental stages.

Small intestine Large intestine

Duo _Jej Ile Ce c Col Re c

Fetus (early) PYY ND PP ND GLP-1 10.12 j=2.04 Fetus (mid) PYY ND PP ND 0.40iOAO 7.60iO.25 0.80 iO.41 ND

5A6i: 1.54 7.20i: I.75

0.53 iO.26 5.00i2.54 0.66iO.35 1.60iO.85 GLP-1 13.33i1.771 8.26j=1.332 ll.6i2,035 Fetus (late) PYY 0.75 j=0.35 PP ND GLP-I 10.53 i 5.27 Calf (su&1ing) PYY ND PP ND

I.33 iO.33 3.56iO.81 I.20iO.60 0.40iO.05 9.06 i2.033 5.23 j=0.89 0.03 iO,03 2.36i 1,07 0.03 iO.03 0A6iOA6 GLP-1 6.00 i 0.70c 2.80 i 0.49 2.43 j=0.95 Calf (weiming) PYY ND 0.06 i 0.06 0.80 j=0.56 PP 0.95iO.03 ND 0.16iO.12 GLP-1 3.73 i 1.25 2.80 j=0.70 I.70 j=0.46 Calf (weane d) PYY ND 0.06 i 0.06 0.76 iO.37 PP 0.33 i 0.17 0.03 i 0.03 0.33 iO.24

GLP-I 3.80iO.96g 2.57i1.18 3.63iO.53h

Adult, PYY ND ND 0.56 i 0.29 PP ND ND 0.06 iO.03 GLP-1 4.96 i 0.98 2.60 j=0.05 3.20 j=0.23 3.86i2.37 4,00i2.06 0.66iO.32 ND 6.66j=2.01 4.40i I.04 5.06i2.53 5.6 i2.91 1.86iO,12 1A6j=0.75 10.27i5.14 0.13 j=0.13 ll.73 j=4.72 16,27 j=1.71a, 9 2.53 iO.31 6.93iO.55 9.46i1.757 15.73j=l.5410 3.06i 1.68 6.13 j=1.52 1A6iO.86 I.33 j=0.88 7.73i2.366 8.93j=2.468 1.30 i 0.73 2.30 j=0.45 0.13 iO.13 0,90iO.32 0.93 iO.32 2.63 iO.20 1.33 i 0.52 1.93 i=0.36 0.10iO.10 0.SOLO.05 0.60iO.45 I.06iO.61 0.96 i 0.29 1.87 j=0.43 0.03 iO.03 0A6iO.08 0.96 i 0.38 2.33 iO.28 2.46iO.29 1.36iO.75 0.36 i 0.08 0.50 iO.40 1,33 iO.08 2.20iO.91 7.80 i=2.27 0.93 i 0,93 7.40 i3.81 3.76 iO.53b 1.00 iO.57 5.66i 1.25d 3.26 iO.14 0.90 iO.58 5.56i2.01e 5.03 i:0.33f 0.83 iO.18 3.77 i 0.50i 4.20 i 2.10j 0.73 iOAO 5.63 i 1.67k

Data represent mean i SEM; Duo: duodenum, Jej:jejunum, Ile: ileum, Col: colon, bc: rectum.

PW: _peptide YY, PP: pancreatic polypeptide, GLP- 1:glucagon-like peptide-1, ND: not detected.

Chapter

3

Quantitative

Immunohistochemical

Study

_{of Endocrine}

Cell

Distribution

in the Bovine

Large

Intestine

3.1.Introduction

The _{gastrointestinal} tract is the largest _endocrine organ in the body

(65).

Gut hormones are _{chemical messengers which} are localized in the

endocrine cells distributed

throughout

the mucosa _{of glandular stomach and}

intestine

(65,

68,

_59).

They are _playing important _roles in _entire digestive

tract to _{regulate many} digestive functions, _Such as _{secretion, absorption and}

motility. Many studies have elucidated the regional distribution and relative frequencies _{of various} kinds _{of endocrine cells} in _{the gastrointestinal} tract _of

different _animals. However, detail _studies on the distribution _{of endocrine}

cells in different parts of the large intestine especially in domestic animals have _not been _{reported yet.} The large intestine, as _wen as _stomach, has

important _{physiological} function in the digestive process of ruminants

(33).

Previous _reports on the _{endocrine cells} in the _ruminant large intestine

examined only few portions

(3,

8, 12,

39).

The discriminative portions, which

are _{characteristic} to the _{spiral colon and} rectum, were _{not examined at} so

clear the detail of endocrine cell distributions in eleven different portions of large intestine _{of cattle.}

3.2. Materials _{and methods}

six calves in two _groups

(suckling

_and

'weaned)

were _used in this study.

The age of the suckling calves were between 5

-7 weeks, and weaned calves

were 7 _months. Tissue _samples were taken from _eleven parts of the large intestine detailed in Chapter 1 _{and processed} for _{paraffin sections} as

described before. The _primary antisera used in this chapter, in addition to

those _used in Chapter 2, were _raised in _{rabbit against chromogranin}

(CG:

1:15,000, Code 20085, INCSTAR, Stillwater, MN,

_USA),

_serotonin

(Ser:

15,000, Code _sero-23, donated by Dr.

_{Nishiitsutsuji-Uwo, Kyoto),}

_and

somatostatin

(son:

I:10,000, Code 20H2T,

INCSTAR).

Immunohistochemical

procedures were _mentioned in Chapter I.

3.3. Results

Six types _{of endocrine cells} were detected _with the antisera against CG,

Ser, GLP- 1, PW, PP _and Son.in _eleven different _{portions oflarge} intestine _of

suckling and weaned calves. The

_regional

distribution and relative frequencies _{of these endocrine cells} were different in _{each parts of large}

between two _{groups, suckling and weaned.} The distribution _{and relative}

frequencies _{of each} immunoreactive _{endocrine cen will} be discussed

individually.

CG-immunoreactive _cens were _abundantly detected in _{an segments of}

the large intestine. The _general distribution _{of CG-immunoreactive cells} was

higher

in _{the most} distal _{parts of the colon and} two _{parts of the rectum}

(Fig

3.

1A-C _and

_3.2A-C).

The frequency _of CG-immunoreactive _cells were

significantly

higher

in the descending colon of both suckling and weaned

groups

(sucklmg:

19.03 i 0.61, weaned: 18.53 i

_3.06),

_{sigmoid colon of both}

groups

(suckling:

22.90 i 0.87, weaned: 18.37 i

2.39),

ampulla of rectum of

both _groups

(suckling:

27.90 i _{1.55, weaned:} 23.37 i

_5.79)

_{and rectum}

_just

cranial to the anorecta1 lme of both groups

(suckling:

21.60 j= 0.86, weaned:

17.40 i

_{0.78) (Fig.}

3.10A, Table

_3.I)

The _general distribution _of Ser-immunoreactive _cens was _almost

similar to that of CG;

higher

in the most distal parts of the colon and two

parts of the rectum

(Fig

3.3A-C, and Fig.

3.4A-C).

Significant differences

(P

<

o.o5).f

Ser-immun.reactive

tells

_{were.bserved in sign.id c.1.n.f sucklmg}

group

(19.33

i

0.87),

ampuna ofrectum of suckling group

(23.37

i

0.81)

and

rectum

just

cranila to the anorecta1 line of both groups

(suckling:

18.77 j= I.03,

weaned: 10.37 i

2.60) (Fig

3. 10B, Table

3.1).

GLP- 1-immunoreactive _cens were _rarely detected in the cecum _and the

distal _{parts of the colon and} two _{parts of the rectum}

(Fig.

3.5A-C _and Fig.

3.6A-C).

No _significant differences _of GLP-1-immunoreactive _cells were

observed between any regions of the large intestine as _well as two different developmental _{stages, suckling and weaned}

(Fig

3. 10C, Table 3.

_1).

The frequencies _of PW-immunoreactive _cells were _{varied among}

regions (Fig.

3.7A-C,

3.8A-C).

They were few in the cecum _and the proximal

portions of the colon, but were _{significantly}

higher

in _{the sigmoid colon}

(4.

10

i

_0.40)

_{and ampuna ofrectum}

(3.60

i

_0.43)

_{of the suckling group}

(Fig.

_3.llA

and Table

3.1).

PP-immunoreactive _cens were detected _rarely in _all portions of the

large intestine.

(Fig 3.9A-C).

The PP-immunoreactive _cells were _very few in

the cecum _{and proximal parts of the colon, increased} in the middle portions,

descending _{c.1.n, Sigm.id c.1.n and ampulla.frectum}

(Fig.

3.llB, Table

6.

_I).

Son-immunoreactive _cells were _only detected _with a _very few _number

in the transverse _{colon of the suckling stage.} They were _not detected in any

other portions of both Sucklmg and weaned groups

(Fig.

3. llC, Table 3.

1).

3.4. Discussion

In chapter 1,_{the regional} distribution _{and relative} frequencies _{of three}

types _{of endocrine} cens, PW-, GLP-I- and PP-immunoreactive cells were

numerous in the large intestine _{of an developmental} groups. This fact leads

the present study to investigate the distribution in more detailed topography

of bovine large intestine. The relative frequencies of CG-, Ser-, GLP-1-, PW-,

PP- _and Son-immunoreactive _{endocrine cells} were _evaluated in _eleven

different _{portions of large} intestine in _order to _{understand the characteristics}

of the tortuous intestine of the ruminant. The distinguishing differences in

distributions _{of these six} types _{of endocrine cens} were found in _{each portions}

of large intestine.

General distribution _{of endocrine cells represented} by

CG-lmmunoreactive _cens indicates the distal _abundance in _the bovine _gut. In

addition, each type of endocrine ceus immunoreactive for Ser, GLP-1, PW,

and PP also showed similar tendency to be more _abundant in the distal

portions. Son-immunoreactive cells were too low in frequency to discuss here.

The present study demonstrated the detail distribution of endocrine

cells along with the tortuous large intestine of ruminant. Previous studies on

the _ruminant have so far examined _{only three portions} in the large intestine

(cecum,

_central flexure _{of the spiral colon,} terminal

_rectum)

_{of cattle}

(39),

sheep

(12),

water buffalo

(8),

and mouse deer

(3).Although

these _results

showed the tendency of the abundance ofendocrine cells in the rectum, itwas

not compared in detail with the rest of the large intestine. The eleven

portions of the intestine examined in the present study are the _peculiar

distinguished the distal distribution _{of endocrine cells} in _the bovine large

intestine. It is suggesting that the huge _numbers of endocrine cells in the

large intestine _{especially distal portions of colon and rectum}

_might

be related

to the

_{physiological}

importance _{of those portions of the} digestive _system in

ruminants. This emphasize again that the dista1-to-proximal feedback

regulation

(59, 89)

may play important roles in the digestive tract of the

ruminant. It iS necessary to make detail

physiological

studies on the lmkage

between different _{portions of the} large intestine _of ruminant and gut

J[ i I > I , I A Q

Fig- 3.I. Chromogranin-immunoreactive _cells in _the large intestine _of

Suckling and weaned calves.

W

cecum _{of the weaned caw,}

(B)

_centripetal

turns _{of colon of the suckling}

_call;

(C)

transvel.Se _{COlon of the weaned calf.}Bar

I 4 JQ. i ∼ I . I r V I i QL /- +-I I I . 1 II ･ _IJ I I I 1

i.

(I t4 I I ･4 .1I I I 1 ･L1 I I L -'I I ∼jt∼ _I I Jl. fr tJ r .;_I ,. F i

Fig. 3.2. Chromogranin-immunoreactive _cells in the large intestine _of

suckling and weaned calves.

W

descending colon in the suckling

calf

(B)

ampuna of rectum in the Suckhg

calf (C)

rectum

just

cranial to the

. I 8 . I I I f I :-piH1

Fig. 3,3. Serotonin-immunoreactive cells in the large intestine of 8uCklmg and

weaned calves.

W

cecum _{of the Sucklmg calf,}

0)

_proximal loop ofcolon in the

weaned

_{calf (C)}

centripetal turns ofcolon in the weaned calf.Bar = ₁₀₀

I

L. -.I. i . -I/ ;:_:.

T.-8

'4 i IA + I J

rA

I . 4I I A o I I 1 I

Fig. 3.4. Serotonin-imnunoreactive cens in the large intestine of suckling and

weaned calves.

W

descending colon of the suckling calf,

0)

ampul1a of

rectum in the Suckling calf,

(C)

rectum

just

cranial to the anorectal he in the

rT-^T{ _{f,I--- A}AIIT--I

:LL: l.'l

i/.A.;/ i

;'ht..i;

'1

-_._.__ _._____

Fig. 3.5. Glucagon-like _peptide- 1-immunoreactive _cells in the large intestine

of suckling and weaned calves.

W

proximal loop of colon in the suckling calf,

0)

transverse _colon in _{the weaned calf,}

(C)

descending _colon in _{the suckling}

rI +

tI:

i.'Jli3-_if,I,;A;ti;

_:i..

;i.

Fig. 3.6. Glucagon-Eke _{peptide-I-immunoreactive} Gens in the large intestine

of suckhg and weaned calves.

W

sigmoid colon in the suckhg calf,

(B)

ampul1a of rectum in the suckhng

_{calf (C)}

rectum

just

cranial to the

Fig. 3.7. Peptide W-inmunoreactive _cens in the large intestine _{Of suckling}

and weaned calves.

W

distal loop of colon in the weaned

call;

(B)

transverse

colon in the suckling calf,

(C)

descending colon in the suckling calf.Bar = loo

T ･/-IF:_--i:#i Ill, ∼y ILL_1{ I (;.:jb A I ∼ ;:T -_i-?TdW>'7Tl ∼ .LL -r..i ･t :,lJ.Y1:-(.:i , L,/.;I i,I .-JL i:. I. .I. - -...--Yt .

i

--.r ;.I -.'(. -I J 1.L 1-. .-.)jp';.; :. I I.:...-.

::,.:..

,:::;.:i"I.33f:

.1L -J! ..)I -T<. A-( I.I

.ti..:

.T. [t.

'r'S.

L '*

:li.

i::;..-il.

I:

Li_

_-,,_ir!ii

4._'./ Tit .I.; > -tl ∼'. ; _:rL,"_..iL_

Fig. 3.8. Peptide YY-inmunoreactive _cells in the large intestine _{of suckling}

and weaned calves.

W

signoid colon in the Suckling calf,

(B)

ampuna of

rectum in the suckling

_calf

(C)

rectum

just

cranial to the anorectal lme in the

weaned calf.Bar = 100

/tti 1 1

LB

y. I

''l

l4?;.=if3

i.I lTpY

8 I -

-'l-:=:>

_. .∼_'1 .)-L :.= ,? ,I-7;

Fig. 3.9. Pancreatic _{polypeptide-inmunoreactive} cells in the large intestine of

suckhg and weaned calves.

W

centrifugal turns Of colon in the weaned calf,

Q})

transverse _{colon of the suckling calf, Bar} = loo

w

40

g30

I-ho

7B

guE

0 1

S

･i

Ta

a

I 2 3 4 5 6 7 8 9 10 ll Large intestinal _portions

d Suckling D Weaned

Fig. 3.10. Distribution _{and relative} frequencies _{of endocrine cells} per unit area

(625 Ltm2)

in the large intestine _{of suckling and weaned} calvesL CG:

chromogranin, Ser: serotonin, GLP-1:

glucagon-like

peptide-1, 1: cecum, 2:

proximal loop of colon, 3: centripetal turns of colon, 4: central flexure of colon, 5: centrifugal turns _{of colon,} 6: diStal loop _{of colon,} 7: transverse _colon, 8: descending _{colon, 9: Sigmoid colon, 10: ampulla of} rectum, ll: rectum

just

(A) 5 4 a =3 1= E} i2 y U I I 2 3 4 5 6 7 8 9 10 I ) 2 3 4 5 6 7 8 9 10 1L Large intestinal porLioTLS

( Sucklitlg a Weaned

Fig. 3.ll. Distribution _{and relative} &equencies _{of endocrine cells} per unit

area

(625 pm2)

in the large intestine Of suckling and weaned calves. PYY: peptide W, PP: pancreatic polypeptide, Son: somatostatin, 1= cecum, 2:

proximal loop of colon, 3: centripetal turns of colon, 4: central nexure of colon,

5: _centrifugal turns _{of colon,} 6: distal loop _{of colon,} 7: transverse _colon, 8:

descending _{colon, 9: sigmoid colon, 10'. ampulla} of rectum, ll: rectum

just