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1. GENERAL AND COMPARATIVE ENDOCRINOLOGY 86, 275-283 (1992)
An lmmunocytochemical Study of Endocrine Gel
in the Early Fetal Guinea Pi
S.REDDY,N. J. BIBBY, AND R. B.ELL~QTT
Department of Pediatrics. School of Medicine, University of Auckland, Private Bag, Auckland, New Zeaiand
Accepted August 29, 1991
The presence of insulin, glucagon, pancreatic polypeptide, and somatostatin containing
cells and their ontogenic changes were investigated immunocytochemically in the early fetal
pancreas of the guinea pig (Days 2.5-40). In the earliest tissues examined (Day 25 and Day
30) brightly staining glucagon cells were the most predominant endocrine cell population,
followed by slightly fewer and weaker staining pancreatic polypeptide cells. Insulin and
somatostatin immunoreactive cells were less numerous. At Day 25 all endocrine cells were
located within the pancreatic tubules where some glucagon cells also coexpressed insulin.
Similar dual immunoreactivity was present at Day 30. At Day 25 some of the pancreatic
polypeptide cells also showed coexpression of somatostatin which persisted until Days
35-40. At these later stages insulin and somatostatin cells were increasingly frequent. Glu-
cagon and pancreatic polypeptide cells were also conspicuous. The four endocrine cell types
were found either in the pancreatic tubules or in the developing islets where they began to
acquire an adult-like topographic distribution. These studies in the fetal guinea pig show that
the four islet hormonal cells cytodifferentiate from an early stage. A small proportion of
endocrine cells coexpress either insulin and glucagon or pancreatic polypeptide and somato-
statin. Q 1992 Academic Press, Inc
The mammalian pancreas develops as an
outgrowth of the fetal gut during early em-
bryology (reviewed by Pictet and Rutter,
197’2) so that ultimate endocrine and exo-
crine components are derived from a com-
mon pool of precursor cells. During this ini-
tial phase of pancreatic organogenesis nu-
merous epithelial cells of the primitive
tubules undergo cytodifferentiation into
specific endocrine cell types and acinar
s (Rutter, 1980). The early chronologi-
cal development of the four major endo-
crine cell types containing insulin, gluca-
gon, pancreatic polypeptide (PP), and so-
matostatin has been studied in a few
mammalian species including the rat,
sheep, pig, and human, and in a marsupial
(opossum) by immunocytochemical means
(Alumets et al., 1983; Krause et al., 1989;
Reddy et al., 1988; Stefan et al., 1983;
Yoshinari and Daikoku, 1982). These onto-
genie studies of early fetal tissue showed
that the development of the endocrine cell
types can be either almost sync~r~~~~s or
chronological with differences in number
and the topographical distribution of the
hormonal cell types as pregnancy proceeds
(see Reddy and Elliott, 1988).
more recent immunocytochemi
in the fetal rat suggested early coexpression
of glucagon and insulin in a p~op~~t~~~ sf
endocrine cells of the developing pancreas
(Hashimoto et al., 1988).This dual colocal-
ization was also reported previously for
glucagon and PP in the adult testudine and
rat pancreas (Kaung, 1985; Rhoten, 1987).
Whether the reported coexpression of islet
hormones in the fetal and adult is more
widespread amon the mammals than pre-
viously thought is unclear.
The guinea. pig appears to have diverged
from other mammals early in their phylog-
eny. In addition, its insulin molecule is
markedly different in primary structure.
275
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Copyright Q 1992 by Academic Press: 8~.
AlI rights of reproduction in any :orm reserved.

2. 276 REDDY, BIBBY, AND ELLIOTT
Studies on the early development of cells
containing insulin, glucagon, PP, and so-
matostatin in the fetal pancreas of this ro-
dent are limited. Histochemical procedures
demonstrated insulin and glucagon cells in
the fetal pancreas of the guinea pig (Peters-
son, 1966). Subsequently, antibodies to in-
sulin, glucagon, somatostatin, and PP al-
lowed immunocytochemical identification
of the corresponding cells in the adult
(Baskin et al., 1984; Reddy et al., 1985;
Watkins et al., 1980). The localization and
topographical distribution of the four major
endocrine cell types in fetal pancreatic tis-
sue of late gestation have been compared
with the condition in the adult pancreas
(Reddy et al., 1985). In view of the limited
information on islet cell development, the
structural distinctness of its insulin, and the
early phylogenetic divergence of this spe-
cies, the ontogeny and distribution of insu-
lin, glucagon, PP, and somatostatin immu-
noreactive cells in the early fetal pancreas
(Day 25-40) of the guinea pig using immu-
nocytochemistry are reported.
MATERIALS AND METHODS
Eight female guinea pigs were remated on the day of
delivery of fetuses from previous pregnancies. Fetuses
from two pregnant animals (two to three fetuses per
mother) were studied at Days 25, 30, 35, and 40 of
gestation (gestation in the guinea pig: 63 days). Fol-
lowing sacrifice of the pregnant animals, the fetal
stomach with the pancreas attached to it ventrally was
excised immediately and fixed in Bouin’s fluid for 2-3
hr. Because the fetal pancreas is extremely soft and
minute prior to these stages, whole fetuses from Day
25 of gestation were removed and the head and caudal
regions trimmed off and fmed as above. All tissues
were then dehydrated through increasing concentra-
tions of ethanol, cleared in chloroform, and embedded
in paraffin (56”). Sections of 4 pm were cut and ad-
hered to gelatinized slides. Selected sections from dif-
ferent levels were stained by hematoxylin and eosin (H
and E) to identify pancreatic tissue for subsequent im-
munocytochemical staining.
Antibodies to bovine insulin (Dr. J. R. Crossley,
Auckland, New Zealand), bovine pancreatic polypep-
tide (anti-BPP serum, Dr. R. E. Chance, Lilly Re-
search Laboratories, Indianapolis, IN), porcine gluca-
gon (Dr. J. E. Livesey, Christchurch, New Zealand),
and somatostatin (Dr. J. R. Oliver, Adelaide, Austra-
lia) and the corresponding antigens were identical to
those used in previous immunohistochemical studies
(Reddy ef al., 1985). The glucagon antiserum used in
this study (M107) shows no cross-reactivity with insu-
lin and BPP and less than 2% cross-reactivity with
glicentin (personal communication, Dr. J. E. Live-
sey). The same antiserum has previously been used by
others for immunocytochemical studies of glucagon
(Ravazzola et a/., 1979; Stefan et a/., 1983). Antibod-
ies to synthetic somatostatin-14 (Bachem, CA) were
raised in rabbits as described previously (Buckerfield
et al., 1981). In radioimmunoassay this antiserum
demonstrated no cross-reactivity or less than 0.01%
cross-reactivity with B-endorphin (61-91), Leu-
enkephalin, Met-enkephalin, glucagon, vasoactive in-
testinal peptide, insulin, C-peptide, human growth
hormone, and substance P (Dr. J. R. Oliver, personal
communication).
Insulin, glucagon, PP, and somatostatin cells in fetal
pancreatic sections were localized by indirect immu-
nofluorescence as previously reported, with minor
modifications (Reddy et al., 1985). Sections were in-
cubated with antibodies to insulin (l:SO), glucagon
(1:50), BPP (l:lOO), and somatostatin (1:50), diluted
with phosphate-buffered saline (PBS) for 18 hr at room
temperature. After the sections were washed in excess
PBS, they were incubated with either sheep anti-rabbit
y globulin or sheep anti-guinea pig y globulin labeled
with fluorescein isothiocyanate (Wellcome Laborato-
ries, United Kingdom) at a dilution of 1:lO in PBS for
1hr at room temperature. After farther washing, sec-
tions were mounted in DABCO reagent and the immu-
nofluorescence was observed with an Olympus fluo-
rescence microscope. After photography of appropri-
ate fields, sections were stained with H and E.
The specificities of the above immunoreactive stain-
ing were tested either by replacing the primary anti-
sera with normal rabbit or guinea pig serum or with
PBS, or by application of the antisera to tissue sec-
tions after absorption with the corresponding antigens
(40-50 pg per milliliter of undiluted antiserum) in the
immunohistochemical procedures.
RESULTS
The main histological features of the
early developing pancreas of the fetal
guinea pig are shown in Fig. 1. Numerous
primitive tubules consisting of epithelial
cells and incompletely differentiated islet
aggregates embedded in a cellular matrix of
densely arranged and scattered nuclei are
prominent at the early stages. In addition,
regions of the immature pancreas can be

3. PANCREATIC HORMONES IN THE FETAL GUINEA PIG 247
FIG. 1. Pancreatic sections from a 30-day (a) and a 35-day (b) guinea pig fetus stained by hematox-
yiin and eosin. Arrowhead, pancreatic tubules; Arrow, islet-like structures. (a) X310, (b) x 155.
seen as an outgrowth of the embryonic gut
and as contiguous distally with the devel-
oping spleen (results not shown).
In the Day 25 fetal pancreas numerous
scattered epithelial cells of the primitive tu-
bules showed positive and usually bright
staiaing for glucagon (Fig. 2). These immu-
noreactive cells were located either singly
or occasionally as “doublets.” Cells show-
ing immunoreactivity to PP had a similar
istribution but were slightly less numerous
and with usually weaker staining. In con-
trast, insulin immunoreactive cells were far
less numerous than glucagon and PP cells
and were located within the tubular epithe-
hum. Densely clustered insulin cells were
observed only occasionally. In serially
stained sections a few cells with strong glu-
cagon staining showed weak coexpression
of insulin (Figs. 2a, 2b). Somatostatin im-
unoreactive cells were least numerous at
ay 25 and were also located within the
tubular cells (Figs. 2d, 2h). In serial sec-
tions, a few cells showing immunoreactivi-
ty against PP as well as somatostatin were
also noted (Figs. 2c, 2d).
At the Day 30 stage the four imm
active endocrine cell types had an overall
distribution similar to that of Day
the glucagon cells persisting as the
inant cell type (Fig. 3). A few cells also
showed coexpression of either insulin
glucagon or PP and somatostatin (Fig.
The four hormonal cell types began to
pear in some of the immature islets,
The distribution of insulin, glucagon,
and somatostatin cells was essentially
ilar at Days 35 and 40 but show
differences from the ea
these stages endocrine
prominent in both the duct
let-like structures (Fig. 4).
age more insulin cells were distributed as
clusters within the developing islets ~Gluca-
gon was expressed in distinct cells usually
located in the islet periphery. Somat~sta~i~
immunoreactivity ap
ber of cells than in t
pancreas, being dist
of the ductules and al
they occurred either
nally. Some cells which showed irnrn~~~~~~

4. 278
FIG. 2. Immunofluorescence localization of ins ulin (a, e), glucagon (b, f), PP (c, g), and somatostatin
(d, h) in pancreatic sections of a 2%day guinea pig fetus. (a, b, c, d) Serial sections; (e, f, g, h) different
areas of the pancreas stained for insulin, glucag on, PP, and somatostatin, respectively. In a and b
arrows point to the same cell coexpressing insul in and glucagon, while in b arrow heads indicate a
“doublet” glucagon cell. In c and d arrows point to cells which coexpress PP and somatostatin. In f
glucagon cells (arrows) are seen closer to the embryonic gut wall (arrow heads). A somatostatin cell
is seen in h with elongated process (arrow). X311
0.

5. acti
cha
h-22
sta
PANCREATIC HORMONES IN THE FETAL GUINEA PHG 279
FIG. 3. Four serial sections stained by immunofluorescence for insulin (a), glucagon (b), PP (c), and
somatostatin (d) from a 30-day fetal guinea pig pancreas. Note numerous and brightly staining giuca-
gon cells in b. In a and b arrows point to cells which coexpress insulin and glucagon while in c and d
they point to cells which coexpress PP and somatostatin. X3IO.
ivity to anti-somatostatin possessed the showed coexpressisn of bot
.racteristic elongate processes. The dis- (Figs. 4c, 4d).
hution of PP ceils was similar to somato- There was an absence of’ ctive
tin. A proportion of cells, however, cells in pancreatic section fetzd

6. REDDY, BIBBY, AND ELLIOTT
guir lea pig when the primary antisera were tions from the BB rat were stained simt
rep1laced by normal rabbit or guinea pig se- neously with anti-insulin and anti-gluts
rum L,PBS, or antisera preabsorbed with ho- sera by immunofluorescence, specific
mol ogous antigens. In addition, it had been calization of glucagon and insulin ceIIs
prel viously shown that when pancreatic sec- observed (Reddy et al., 1986a).
FIG. 4. Immunofluorescencelocalization of insulin (a), glucagon (b), PP (c). and somatostatin (d) in
pancreatic sections of a 40-day guinea pig fetus. (a and b) Serial sections with arrow heads pointing to
the same islets showing insulin and glucagon cells (arrows); (c and d) serial sections with arrows
pointing to a subpopulation of cells coexpressing PP and somatostatin. x 310.
alta-
tgon
: lo-
was

7. PANCREATIC HORMONES IN THE FETAL GUINEA PIG
DISCUSSION
This study examined the ontogeny and
immunolocalization of insulin, glucagon,
PP, and somatostatin in the early fetal pan-
creas of the guinea pig. The guinea pig pos-
sesses certain unique biochemical, devel-
opmental, and reproductive features com-
pared with other rodents such as rats and
mice. For example, the gestational period
in the guinea pig is considerably longer (63
days versus 21 days in rats and mice) and its
newborn is much more mature. Insulin, glu-
cagon, and vasoactive intestinal peptide
from this species show important amino
acid substitutions when compared with the
corresponding peptides from other mam-
mals (Du et aI., 1985; Huang et al., 1986;
Smith, 1966). In addition, the pancreatic l3
cell of the guinea pig is resistant to the di-
abetogenic agent, alloxan, and may possess
regenerating properties following damage
by p cell cytotoxins (Gorray et al., 1990).
The immunocytochemical studies show
that by Day 25 all four hormonal cell types
are present in the fetal pancreas but with
notable differences in their relative num-
ber, intensity of staining, and proportion of
cells showing coiocalization. At this stage,
strongly staining glucagon cells were the
most numerous endocrine cell type, fol-
lowed by PP and insulin cells. Only a few
scattered somatostatin cells were observed.
In the newborn opossum, glucagon and PP
s have also been reported recently as
the most numerous endocrine cell types
(Krause et al., 1989). The coexistence of
the four distinct cell populations in the
early pancreas of human, pig, and sheep
has also been reported with some differ-
ences in the relative numbers of the cells
(Alumets et al., 1983; Reddy et al., 1988;
Stefan et al., 1983). For example, in the
human fetus of 8 weeks gestation, a size-
able proportion of the endocrine cells
stained for insulin and glicentin with gluca-
gon and PP cells being rare (Stefan et al.,
$3). These data in the early human differ
from the present observations in guinga
pig. There are also some differe s com-
pared with the fetal pig, in which i~s~li~~
glucagon, and somatostatin cells are
present by 4 weeks but PP cells a
(10 weeks, Alumets et al., 1983).
however, the first endocrine cell t
cells appear at Days 14 and 17) r
(Yoshinari and Daikoku 1982).
sheep of early gestation (Day 4
is a predominance .of insulin and gluca
cells accompanied by isolated PP and
matostatin cells in the primi
(Reddy et ad., 1988). The significance
the predominance of glucagon cells in
early fetal pancreas of the guinea pig is un-
clear.
In the 35 to 40-day fetal guinea
cress all four endocrine cell types increase
in number within the pancreatic d~~t~la~
cells and in the developing islets weeks they
begin to acquire a topographical mistrial-
tion similar to those in late gestation and in
adult tissue (Reddy et al., 1985). Tksis in-
crease in endocrine cell populations also
occurs in the fetal pancreas of most other
species examined, although accurate mor-
ata are lacking in some
ous studies (Reddy and Elliott, ~9~$).
present study the appearance of elon
processes emanating from some of the
cells are notable. These proces
a feature of D cells from several
malian pancreas where they may act in a
paracrine fashion (Watkins et al., I
Reddy et al., 1986b).
ent study, the coexpression of
ucagon in a few cell aYs
and somatostatin at 25--
40 is noteworthy. These observations are
not surprising since the mature e
ment. The co
influence of revelatory ele

8. 282 REDDY, BIBBY, AND ELLIOTT
to cytodifferentiation of endocrine cell
types elaborating unique peptides. Re-
cently, Hashimoto et al. (1988) demon-
strated the coexpression of insulin and glu-
cagon in a proportion of pancreatic cells
from Day 12.5 to Day 20.5 in the fetal rat.
The colocalization of PP and somatostatin
observed in the guinea pig has not been re-
ported in other species. However, the co-
existence of glucagon and PP within the
same islet cell has been shown in the adult
rat and tortoise (Kaung, 1985; Rhoten,
1987). The antisera used in the present im-
munocytochemical studies have previously
been shown to be specific for the corre-
sponding antigens (Ravazzola et al., 1979;
Buckerfield et al., 1981; Reddy et al.,
1986a,b). Moreover, the anti-BPP serum
used is from the same source as that used
by Rhoten and Kaung (Dr. R. E. Chance,
Eli Lilly). The dual colocalization is there-
fore likely to be genuine but requires con-
firmation by other techniques such as in
situ hybridization or double-labeling immu-
nohistochemistry.
ACKNOWLEDGMENTS
We thank Ms. Omie Wijeyesinghe for typing the
manuscript and Mr. V. Jensen for the pregnant guinea
pigs. We are grateful to Drs. R. E. Chance, J. R. Ol-
iver, J. E. Livesey, and J. R. Crossley for the anti-
sera. This study was financed by the National Chil-
dren’s Health Research Foundation of New Zealand.
REFERENCES
Alumets, J., Hakanson, R., and Sundler, F. (1983).
Ontogeny of endocrine cells in porcine gut and
pancreas: An immunocytochemical study. Gus-
troenterology 85, 1359-1372.
Baskin, D. G., Gorray, K. C., and Fujimoto, W. Y.
(1984). Immunocytochemical identification of
cells containing insulin, glucagon, somatostatin
and pancreatic polypeptide in the islets of Lang-
erhans of the guinea pig pancreas with light and
electron microscopy. Anat. Rec. 208, 567-578.
Bucker-field, M., Oliver, J., Chubb, I. W., and Mor-
gan, I. G. (1981). Somatostatin-like immunoreac-
tivity in amacrine cells of the chicken retina. Neu-
roscience 6, 689-695.
Du, B.-H., Eng, J., Hulmes, J. D., Chang, M., Pan,
Y. C. E., and Yalow, R. S. (1985). Guinea pig has
a unique mammalian VIP. Biochenr. Biophys.
Res. Commun. 128, 1093-1098.
Gorray, K. C., Maimon, J., and Schneider, B. S.
(1990). Regeneration of islet 8 cells in the alloxan-
treated guinea pig. In “Lessons from Animal Di-
abetes.” [Abstract]
Hashimoto, T., Kawano, H., Daikoku, S., Shima, K.,
Tanaguchi, H., and Baba, S. (1988). Transient co-
appearance of glucagon and insulin in the progen-
itor cells of the rat pancreatic islets. Anat. Em-
bryol. 178, 489-497.
Huang, C. G., Eng, J., Pan, Y. C. E., Hulmes, J. D.,
and Yalow, R. S. (1986). Guinea pig glucagon dif-
fers from other mammalian glucagons. Diabetes
35, 508-512.
Kaung, H.-L. C. (1985). Electron microscopic immu-
nocytochemical localization of glucagon and pan-
creatic polypeptide in rat pancreas: Characteriza-
tion of a population of islet cells containing both
peptides. Anut. Rec. 212, 292-300.
Krause, W. J., Cutts, J. H., Cutts, J. H., and Ya-
mada, J. (1989). Immunohistochemical study of
the developing endocrine pancreas of the opos-
sum (Didelphis virginiana). Actu Anut. 135, 84-
96.
Petersson, B. (1966). The two types of alpha cells dur-
ing the development of the guinea pig pancreas. 2.
Zellforsch. 75, 371-380.
Pictet, R., and Rutter, W. J. (1972). Development of
the embryonic endocrine pancreas. In “Hand-
book of Physiology” (D. F. Steiner and N.
Freinkel, Eds.), Sect. 7, Vol. 1, pp. 25-66. Am.
Physiol. Sot. Washington, DC.
Ravazzola, M., Siperstein, A., Moody, A. J., Sundby,
F., Jacobsen, H., and Orci, L. (1979). Glicentin
immunoreactive cells: their relationship to gluca-
gon-producing cells. Endocrinology 105, 499-508.
Reddy, S. N., Bibby, N. J., and Elliott, R. B. (1985).
Cellular distribution of insulin, glucagon, pancre-
atic polypeptide hormone and somatostatin in the
fetal and adult pancreas of the guinea pig: A com-
parative immunohistochemical study. Eur. .7.Cell
Bid. 38, 301-305.
Reddy, S., Bibby, N. J., Fisher, S. L., and Elliott,
R. B. (1986a). Longitudinal study of lirst phase
insulin release in the BB rat. Diubetologiu 29,
802-807.
Reddy, S., Bibby, N. J., Fisher, S. L., and Elliott,
R. B. (1986b). Immunolocalization of insulin, glu-
cagon, pancreatic polypeptide and somatostatin in
the pancreatic islets of the possum, Trichosurus
vulpecula. Gen. Comp. Endocrinol. 64, 157-162.
Reddy, S., Bibby, N. J., and Elliott, R. B. (1988). An
immunofluorescent study of insulin-, glucagon-,
pancreatic polypeptide- and somatostatin-

9. PANCREATIC HORMONES IN THE FETAL GUINEA PI@
containing cells m the early ovine fetal pancreas.
Q. J. Exp. Physiot. 73, 225-232.
Reddy, S., and Elliott, R. B. (1988). Ontogenic devel-
opment of peptide hormones in the mammalian
fetal pancreas. Experientia 44, 1-9.
Rhoten, W. B. (1987). Co-localisation of glucagon and
pancreatic polypeptide in testudine pancreas. Ex-
perientia 43, 428-430.
Rutter, W. J. (1980). The development of the endo-
crine and exocrine pancreas. In “The Pancreas”
(P. J. Fitzgerald and A. B. Morrison, Eds.), pp.
30-38. Williams & Wilkins, Baltimore, London.
Smith, L. F. (1966). Species variation in the amino
acid sequence of insulin. Am. /. Med.
666.
Stefan, Y., Grasso, S., Perrelet, A., and Orci, L.
(1983). A quantitative immunofluorescent study
of the endocrine cell populations in the ~eve~~~i~~
human pancreas. Diabetes 32, 293-301.
Watkins, W. B., Bruni, J. F., and Yen, S. S. C.
(1980). B-Endorphin and somatostatin in the pan-
creatic D-cell coiocalization by
chemistry. J. Histochem. Cytoch
1174.
Yoshinari, M., and Daikoku, S. (1982). Gntogenetic
appearance of immunoreactive endocrine cells in
rat pancreatic islets. Anat. EmbryoE. 165, 63-40.