Báo cáo khoa học: " Expression of pituitary adenylate cyclase activating polypeptide and its type I receptor mRNAs in human placenta" doc

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J. Vet. Sci.
(2005),
/
6
(1), 1–5
Expression of pituitary adenylate cyclase activating polypeptide and
its type I receptor mRNAs in human placenta
Phil-Ok Koh
1
, Chung-Kil Won
1
, Hae-Sook Noh
2
, Gyeong-Jae Cho
2
, Wan-Sung Choi
2,
*
1
Department of Anatomy, College of Veterinary Medicine and Institute of Animal Medicine, Gyeongsang National University,
Jinju 660-701, Korea
2
Department of Anatomy and Neurobiology, College of Medicine, Institute of Health Sciences, Gyeongsang National University,
Jinju 660-751, Korea
Pituitary adenylate cyclase activating polypeptide
(PACAP) was first isolated from ovine hypothalamus and
was known to stimulate the release of growth factor in
various cells. Recently, we reported the cellular

localization of PACAP and its type I (PAC
1
) receptor in rat
placenta during pregnancy. Placenta is a critical organ
that synthesizes several growth factors and angiogenic
factors for the fetal development and its own growth.
However, there is little information regarding the cellular
localization of PACAP and its receptor in human placenta
at various gestations. The aim of the present study was to
define the expression and distribution of PACAP and
PAC
1
receptor mRNAs in the human placenta during the
pregnancy period. PACAP and PAC
1
receptor mRNAs
were expressed in stroma cells of stem villi and terminal
villi. At the early stage, on 7 and 14 weeks, PACAP and
PAC
1
receptor genes were moderately expressed in stroma
cells surrounding the blood vessels within stem villi. These
genes were strongly expressed in stroma cells of stem villi
and terminal villi on 24 and 38 weeks. The expression of
these genes was increased as gestation advanced, and
localized in the same areas. Localization of PACAP and
PAC
1
receptor demonstrate the evidence that PACAP may
play an important role, as an autoregulator or

pararegulator via its PAC
1
receptor. In conclusion, our
findings strongly suggest that PACAP may have a critical
role in physiological function of the placenta for
gestational maintenance and fetal growth.
Key words:
PACAP, receptor, placenta, human
Introduction
The placenta is an essential organ for the fetal
development and the maintenance of pregnancy. It is known
that placenta synthesis the growth hormone [21] and several
growth factors, such as basic fibroblast growth factor and
insulin like growth factor [6,29]. Also, placenta produces
placenta growth factor (PlGF) and vascular endothelial
growth factor (VEGF) [6,29], which are critical factors for
the placental growth and fetal development. As an important
regulator of angiogenesis, VEGF contributes to the
development and growth of the endothelium during the
tissue growth [5,32]. Also, another member of the VEGF
family, PlGF, promotes endothelial cell proliferation in vitro
[16]. The previous study showed pituitary adenylate cyclase
activating polypeptide (PACAP) stimulates the release of
VEGF and acts as a trophic factor in various cells [7,8,17,31,
33]. PACAP has considerable homology with vasoactive
interstinal peptide (VIP) and growth hormone releasing
hormone [17,31,33]. Recently, it was reported that PACAP
and PACAP receptor are present in both the human and rat
placenta at term [23]. Also, even more recently, we reported
the cellular localization of PACAP and PACAP type I

(PAC
1
) receptor in the rat placenta during pregnancy [14].
Therefore, the existence of PACAP in placenta suggests that
PACAP affects placental function.
PACAP was originally isolated from ovine hypothalamus
and was known to stimulate the production of cAMP in
anterior pituitary cells [18]. PACAP exists in two biologically
active forms, PACAP 38 and PACAP 27, sharing the same
N-terminal 27 amino acids [19]. PACAP binds to three type
Ireceptors. Among these receptors, PAC
1
receptor has high
affinity with PACAP 38 and PACAP 27, very low affinity
with VIP [12,28]. But, VIP1 and VIP2 receptors have
approximately equal high affinity for PACAP 38, PACAP
27, and VIP [11,12,28]. PACAP and its receptor have been
found in the central nervous system and its peripheral
tissues, including the hypothalamus, pituitary gland, adrenal
*Corresponding author
Tel: 82-55-751-8716; Fax: 82-55-759-0779
E-mail:
2 Phil-Ok Koh
medullar, testis, and ovary [2,3,10,25,27]. The presence of
PACAP in the hypothalamus, pituitary, and gonads suggests
its roles in the reproductive system. However, the existence,
localization of PACAP and PAC
1
receptor genes in human
placenta at various gestations has been unknown. Thus, the

present study was performed to determine the distribution of
PACAP and PAC
1
receptor mRNAs in human placenta.
Materials and Methods
Tissue preparation
Human placental tissue from legal abortions aged between
6-7 weeks post menstruation (pm) were collected from
normal pregnancies by curettages. Second trimester placenta
from 14-24 weeks of gestation were obtained from induced
abortion of healthy pregnancies and term placenta (38-41
weeks pm) by caesarian section or normal delivery. For
in
situ
hybridization studies, tissues were fixed with 4%
paraformaldehyde in 0.1M phosphate buffered saline (PBS)
and cryoprotected with 20% sucrose phosphate buffer for 24
hr. Placental sections were cut in a cryomicrotome at a
thickness of 15
µ
m, mounted on the Probe-on slides (Fisher
Scientific, USA), and stored at
−
70
o
C. Slides from each
placenta were stained with hematoxylin and eosin for
general morphological observation.
In situ
Hybridization

All solutions were made with sterile water and glassware was
autoclaved to prevent contamination by RNase.
In situ
hybridization histochemistry was carried out, as described by
Angerer
et al
. [1]. Briefly, the slides were dried, washed with
0.1 M PBS, and treated proteinase K, TE buffer, and an
acetylation solution. Sections were covered with prehybridization
buffer containing 50% deionized formamide and incubated at
37
o
C for 1 hr. After removal of the prehybridization buffer, the
slides were covered with the mixture containing the
prehybridization buffer, 50
µ
g/ml yeast tRNA, 10 mM
dithiothreitol, and
35
S-labeled PACAP cRNA probe or PAC
1
receptor cRNA probe [13]. The slides were then covered with
cover glasses and incubated at 60
o
C for 24 hr.
35
S-UTP-labeled
probes were prepared using
in vitro
transcription kit (Promega,

USA). Antisense and sense cRNA probes were purified with a
Sephadex G-50 nick column (Pharmacia Biotech, Sweden) and
eluted with SET buffer (0.1% SDS, 1 mM EDTA, 10 mM Tris,
and 10 mM DTT). Tissue slides were posthybridized in a
posthybridization buffer. Following a wash in 4
×
SSC for 30
min, the sections were then treated with ribonuclease A (50
µ
g/
ml) at 37
o
C for 10 min, washed twice in 2
×
SSC and 1
×
SSC,
transferred to a wash buffer containing 0.1
×
SSC at 65
o
C for
30 mins, and dehydrated in alcohol solutions with ascending
concentrations. Slides were exposed to
β
-max hyperfilm
(Amersham, Sweden) for 4 days in light-tight cassettes at
−
70
o

C, and were dipped into NTB2 emulsion (1 : 1 dilution,
Eastman Kodak, USA), exposed at 4 for 2 weeks, developed in
Kodak D19 developer (1 : 1 dilution, Eastman Kodak, USA) at
15
o
C, and counterstained with hematoxylin. The slides were
observed under a dark and a bright field microscope, and
photographed.
Results
The present study showed the expression and distribution
of PACAP and PAC
1
receptor mRNAs in the human placenta
at various gestations.
In situ
hybridization revealed the
expression of PACAP and PAC
1
receptor mRNAs in stem
villi and terminal villi. Positive cells of PACAP mRNA were
detected in stroma cells surrounding the blood vessels
within stem villi on 7 weeks (Figs. 1A & 2A). PACAP
mRNA was expressed in stroma cells of stem villi on 14
week (Figs. 1B & 2B). Furthermore, positive signals of
PACAP mRNA were strongly observed in the stroma cells
of stem villi and terminal villi on 21 and 38 weeks (Figs. 1C,
1D, 2C & 2D). Signals for PACAP mRNA in these cells
were gradually increased as gestation advanced. However,
PACAP mRNA was very weakly expressed in cytotrophoblast
cells and syncytiotrophoblast cells. There were no detectable

signals in negative control with a sense probe (Fig. 1E).
The hybridization of adjacent sections with PACAP and
PAC
1
receptor cRNA probes showed that PACAP and PAC
1
receptor mRNAs were expressed in the same areas. Positive
signals of PAC
1
receptor mRNA were detected in stroma
cells of stem villi on 7 and 14 weeks (Figs. 3A, 3B, 4A &
4B). PAC
1
receptor mRNA was strongly expressed in stroma
cells of stem villi and terminal villi on 21 and 38 weeks
F
ig. 1. Dark-field photomicrographs of PACAP mRNA expressi
on
i
n the human placenta from 7 (A), 14 (B), 24 (C), and 38 (D
)
w
eeks gestation by
in situ
hybridization. A and B: Positive signa
ls
w
ere moderately observed in stroma cells of stem villi. C and
D:
P

ositive cells were strongly detected in stroma cells of stem vi
lli
a
nd terminal villi. E: No positive signals were detected in negati
ve
c
ontrol with a sense probe. Bar = 200 µm.
PACAP and its type I receptor in human placenta 3
(Figs. 3C, 3D, 4C & 4D). As similar like to the expression
pattern of PACAP, positive signals for PAC
1
receptor in these
cells became strong as gestation advanced. But, PAC
1
receptor mRNA was very weakly expressed in cytotrophoblast
cells and syncytiotrophoblast cells. No positive signals of
PAC
1
receptor mRNA was detected in negative control with
a sense probe (Fig. 3E).
Discussion
In the present study, we showed the cellular localization of
PACAP and PAC
1
receptor mRNAs in human placenta on 7,
14, 24, and 38 weeks. Recently, we reported evidence that
PACAP and PAC
1
receptor mRNAs were expressed in
decidual cells, chorionic vessels, and stroma cells of

chorionic villi in the rat placenta [14]. Also, Scaldaferri
et al.
[23] demonstrated the presence of PACAP and PAC
1
receptor in both the human and the rat placenta at term,
using RT-PCR and immunohistochemistry techniques. They
showed the expression of PACAP at term placenta, but they
did not offer any information as to the distribution of
PACAP and PAC
1
receptor in human placenta at various
gestations, during pregnancy. In this study, we utilized
in situ
hybridization to determine the existence of PACAP and
PAC
1
receptor mRNAs in human placenta. Our data showed
the expression of PACAP and PAC
1
receptor mRNAs in
stroma cells of stem villi and terminal villi. As gestation
advanced, the expression of PACAP and PAC
1
receptor
mRNAs was increased in these cells.
It is known that placenta produces several growth factors
such as insulin like growth factor and basic fibroblast growth
factor. Especially, VEGF and placenta growth factor (PlGF),
which are essential factor for the placental growth and fetal
development [5,32]. VEGF was expressed in stroma cells

within villi in human placenta [26]. Localization of VEGF
in stroma cells demonstrates that VEGF play an important
role in the physiological growth and function of the vascular
system in the villous stroma. Furthermore, PACAP that acts
as a growth factor in various cells, stimulates VEGF release
F
ig. 2. Bright-field photomicrographs of PACAP mRN
A
e
xpression in the human placenta from 7 (A), 14 (B), 24 (C), a
nd
3
8 (D) weeks gestation by
in situ
hybridization. A and B: PACA
P
m
RNA was strongly expressed in stroma cells of stem villi.
C
a
nd D: Positive signals were strongly detected in stroma cells
of
s
tem villi. Arrows indicate the positive cells. Bar = 20
µ
m.
F
ig. 3. Expression of PAC
1
receptor mRNA in the hum

an
p
lacenta from 7 (A), 14 (B), 24 (C), and 38 (D) weeks gestati
on
b
y
in situ
hybridization. A and B: Positive signals we
re
m
oderately observed in stroma cells of stem villi. C and
D:
P
ositive cells were strongly detected in stroma cells of stem vi
lli
a
nd terminal villi. E: No positive signals were detected
in
n
egative control with a sense probe. Bar = 200
µ
m.
F
ig. 4. Localization of PAC
1
receptor mRNAs expression in t
he
h
uman placenta from 7 (A), 14 (B), 24 (C), and 38 (D) wee
ks

g
estation by
in situ
hybridization. A and B: PAC
1
receptor mRN
A
w
as strongly expressed in stroma cells of stem villi. C and
D:
P
ositive signals were strongly detected in stroma cells of ste
m
v
illi. Arrows indicate the positive cells. Bar = 20
µ
m.
4 Phil-Ok Koh
[7]. Also, VEGF has been known to act directly on vascular
endothelial cells by promoting cell proliferation and
permeability. In this study, PACAP was strongly expressed
in stroma cells of stem villi and terminal villi. PACAP
mRNA was localized in the stroma cell surrounding the
blood vessel on 14 and 24 weeks. Also, PACAP mRNA was
expressed in the whole stroma cells on 38 weeks.
Furthermore, PACAP 38 immunostaining was detected in
stroma cells of stem and terminal placental villi [23]. Both
PACAP and VEGF were expressed in stroma cells of
terminal villi. Thus, our data suggest that PACAP stimulates
the release of VEGF and promotes the growth of placenta.

In the previous studies, VIP/PACAP neuropeptide family
regulates the blood flow and hormone secretion in human
placenta [4,9,30]. Steenstrup
et al.
[30] reported that PACAP
were expressed in the uteroplacental unit, where it causes a
concentration dependent relaxation on stem villous and
umbilical cord arteries. These results suggest that PACAP
mediates the placental growth and fetal development during
the pregnancy period.
In the previous studies, many researchers demonstrated
that PACAP acts an autocrine and/or paracrine regulator in
various tissues, including ovarian granulose cells, testicular
Leydig cells, and placental tissue [15,20,22,24]. Also, in our
results, PACAP and PAC
1
receptor mRNAs were expressed
in the same areas. Although our data did not elucidate the
physiological role and action mechanism of PACAP in
human placenta, the localization of PACAP and its PAC
1
receptor in the same areas strongly suggest that PACAP may
act as an autoregulator or pararegulator via its PAC
1
receptor
in stem villi and terminal villi during pregnancy. In
conclusion, our findings suggest that PACAP may have an
important role in physiological function of the placenta for
gestational maintenance and fetal growth.
Acknowledgments

This work was supported by the Brain Korea 21 Project,
the Ministry of Education of Republic of Korea, and was
partly supported by the grant from the Korea Science and
Engineering Foundation (KOSEF R04-2003-000-10062-0).
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