Immunohistochemical assessment of ghrelin, GHS

Archives of Perinatal Medicine 13(3), 44-48, 2007
ORIGINAL PAPER
Immunohistochemical assessment of ghrelin, GHS-R, GH, GHRH
and somatostatin in pituitary glands of human fetuses
ANDRZEJ KĘDZIA1, MONIKA OBARA-MOSZYŃSKA1, MARIA CHMIELNICKA-KOPACZYK2
Abstract
In our paper we have assessed with the use of immunocytochemistry the location of ghrelin GH, GHS-R, GHRH and somatostatin. Our studies
suggest that fetal pituitary gland somatotropes between 18-37 week of gestation express GH, ghrelin and GHS-R. These data suggest that
the release of GH in fetal pituitaries might be mediated by ghrelin based on auto- or paracrine action. In the fetal life the hypothalamic
pituitary axis is inactive and the process of its activation most probably begins in the first week of the extrauterine life.
human fetuses, ghrelin, GH, GHS-R, GHRH, somatostatin
Key words:
Introduction
Ghrelin, a 28 acylated amino acid, was for the first time
isolated from the rat’s stomach mucosal lining in 1999 by
Kojima et al. [1]. Various functions were attributed to this
peptide, including the role in the release of growth hormone
(GH) from the pituitary gland and the control of the energetic
homeostasis that regulates the energy usage by influencing
the appetite – the pituitary-intestine axis [2]. The presence of
ghrelin was shown in numerous peripheral organs of the mature human organism and during the intrauterine ontogenesis
[3]. Our earlier papers and the works of other authors have
shown the expression of ghrelin and its receptor in multiple
fetal organs, however the role ghrelin plays in this period of
intrauterine life is not fully appreciated [4]. It is said to have
a role in the differentiation and maturation processes of the
fetal organs. However the expression of ghrelin in pituitary
glands of human fetuses was seldom evaluated, usually during
studies of ghrelin expression in endocrine tumors [5].
The aim of the paper was the assessment of ghrelin expression in somatotropes of human pituitary gland during the
fetal ontogenesis processes and its probable role in this period
of life. Also we wanted to establish the possible correlation
between location of ghrelin and growth hormone. We also
investigated whether there is any connection between pituitary gland and somatotropes in this period of life.
Material and methods
The study included 10 pituitary glands collected during
autopsy from fetuses at different times of the intrauterine life.
The fetal characteristics of the studied group are depicted in
the table 1. The pituitary glands were fixed in buffered formalin and cut in longitudinal and transversal sections with careful
marking of each section. Every 7th section was stained with haematoxylin and eosin, while the rest had immunohisochemistry
for the presence of ghrelin, GH, GHRH, somatostatin and
ghrelin receptor (GHS-R). The data regarding the antibodies
used are shown in table 2. Somatotropes were stained using
anti-GH antibodies.
1
2
Table 1. The characteristics of studied fetuses including week
of gestation, body mass, duration of extrauterine life
Pathology Week of
Duaration
# specimen gestation
Body mass of extrauterine life
number
(hours)
1
8695
18
–
–
2
8732
26
980
7
3
8737
27
910
4
4
8653
31
2800
79
5
8719
32
1320
4 days
6
8629
33
2200
3
7
8652
35
2900
40
8
8721
35
2000
12
9
8656
36
2600
19
10
8669
37
3115
15
#
1
2
3
4
5
Table 2. The list of antibodies used, manufacturers
and the dilution
Polyclonal
Manufacturer
Dilution
antibody
Santa Cruz
Ghrelin
Biotechnology, Inc 1:150
Santa Cruz
GHS-R
Biotechnology, Inc 1:20
GH
DAKO
Ready to use
Santa
Cruz
GHRH
Biotechnology, Inc 1:50
Somatostatin
DAKO
Ready to use
To assess whether ghrelin expression is localized in somatotropes there were two reaction conducted on the same
section; first the ghrelin reaction by incubation of the section
with specific antibody that was left in the fridge throughout
the night, while in the morning the reaction was ended by
enzymes – LSAB+System HRP and subsequent reaction for
GH was performed with the use of EnVision kit using AEC. In
this setup ghrelin was stained with different shades of brown,
while GH was stained red. Cells containing both studied proteins had red-brown color with the red color being more dominant.
II Department of Paediatrics, Medical University in Poznań, Poland
Department of Biochemistry and Pathology, Medical University in Poznań, Poland
Immunohistochemical assessment of ghrelin, GHS-R, GH, GHRH and somatostatin in pituitary glands of human fetuses
Results
Ghrelin
Already in the pituitary gland of the youngest fetus from
18th week of gestation there were scattered or nested ghrelinpositive cells placed mostly in peripheral lateral fields of the
glandular portion of the pituitary gland. Immunopositive
reaction was localized in the cytoplasm, while nuclei were
negative for staining. In the pituitary gland of 26th week old
fetus the number of immunostained cells increased markedly,
and were seen mostly in nests, while their location was similar
as in the 18th week old fetus. The ghrelin expression in cells
differed, ranging from dark brown, through reddish to yellowish (Fig. 1).
45
In the nodular portion with the glandular vesicles makeup the
cells staining positive for ghrelin were few and deposited in
the epithelial cells systems. In the glandular portion of the
pituitary glands coming from the 35-37th week old fetuses we
have found no discernible difference compared to the once
from 31-33rd week except for the increased number of ghrelin
positive cells. The level of expression of ghrelin was similar in
pituitary glands from different periods of fetal life and manifested as brown to reddish-yellow.
GHS-R
Immunoreaction for the ghrelin receptor was localized in
ghrelin positive cells with various strength of staining. In
many cells the reaction appeared as small granulations deposited in the cell membrane. The number of cells rich in receptors was increasing with the increase of the gestational
age.
Growth hormone
The cells with positive reaction for the anti-GH antibody
had similar location to ghrelin-positive ones. There was
staining in the cytoplasm, while nuclei remained negative. In
the earliest weeks, 18-26th the number of GH positive cells
was higher compared to ghrelin-positive cells, while in older
fetuses, 31-35th the difference disappeared (Fig. 3).
Fig. 1. Location of ghrelin in the cells of the glandular portion
of the pituitary gland from the 35 week fetus.
Immunopositive reaction in cytoplasm, negative nuclei
In the pituitary glands from the 31-33rd week old fetuses the
zones of location of the ghrelin-positive cell were more defined. The lateral fields containing a large number of cells
immunostained for ghrelin were divided by a streak of smaller
cells that took up a space between intermediate portion of the
pituitary gland and anterior glandular field. In this region
there were no ghrelin positive cells (Fig. 2).
Fig. 3. The location of GH in the cells of the glandular portion
of the 32 week old fetus. Immunopositive reaction localized
in cytoplasm, negative in nuclei
Fig. 2. The photo depicts a zone with absent reaction for ghrelin,
GH and GHS-R. On the left side of the photo there is a visible
intermediate zone of the pituitary gland, marked with the arrow
The GH positive cells were not encountered in the negative
for ghrelin zones, and also were not present in the nodular
portion of the pituitary gland. For evaluation whether ghrelinpositive cells belong to a family of somatotropes a double
staining reaction was performed on the same sections, with
respect to ghrelin and GH. Most of the cells stained red, while
in cells with strong ghrelin expression on the reddish hue
there was also brown reaction present. The histological analysis regarding the arrangement of cells and their color
confirmed that ghrelin is localized in somatotropes (Fig. 4).
46
A. Kędzia, M. Obara-Moszyńska, M. Chmielnicka-Kopaczyk
Discussion
Fig. 4. The photo depicts the cells stained for ghrelin and GH
in the pituitary gland from 35 week old fetus. On the brown
staining (ghrelin) there is superimposed red staining from GH
GH releasing hormone and somatostatin
In all 10 pituitary glands there was no positive reaction in
the neurohypophysis for somatostatin. In 9 cases there was
also no reaction for GHRH, while in one pituitary gland
coming from a newborn that survived 4 days there was positive immunoreactions for GHRH with a pattern resembling
a delicate net with minute densities in the stalk and neural
portion of the pituitary gland. In the controls there was no
staining (Fig. 5a, b).
Fig. 5 a) Positive immunoreaction for GHRH in the neural portion
of the pituitary gland of the 32 week old fetus, that has survived
for days after birth. There is visible delicate net-like brown staining. b) Negative control with omission of the anti-GHRH antibody
Growth hormone releasing hormone (GHRH) and somatostatin are two classic hypothalamic stimulators and inhibitors that regulate the pulsatile secretion of GH from the
pituitary gland. The role of ghrelin in above mentioned process is not fully elucidated. Rindi et al. based on the available
literature concluded that ghrelin is not regulated by GH in
a way of classic feedback mechanism [3]. A particularly strong
reaction for ghrelin and its receptor was shown based on
immunocytochemistry both in hypothalamus and pituitary
gland. However number of authors question whether ghrelin
detected in the central nervous system is synthesized there;
the latest data based on RT-PCR methods did not detect
ghrelin mRNA in neither the cortex or hypothalamus of
a monkey’s brain [6, 7]. However the RT-PCR detected ghrelin
mRNA in the pituitary; it has been ascribed a role in the
modulation of GH secretion [8-11]. Malagon et al. showed
a direct influence of ghrelin on the release of GH in somatotropes in vitro [12]. Among other things it increases the amount
of free calcium in these cells. Broglio et al. on the other hand
noted that ghrelin does not play a major role in physiologic
control of somatotropes, because there is no modification of its
concentration in the blood after glucagon challenge test [13].
The studies on ghrelin in the pituitary gland of human
fetuses are particularly scant, and this problem was addressed
only while expression of ghrelin was studied in the endocrine
tumors of the pituitary gland [5]. In our paper we have shown
a immunopositive reaction for ghrelin and GH in somatotropes
in a youngest pituitary gland extracted from 18th week old
fetus. Waters and Kaye in the literature review about the role
of GH in the development of the fetus state, that fetal pituitary
synthesizes the GH even earlier, at the end of the first trimester. Apart from presence of GH and detected by us
ghrelin, the fetal pituitary gland somatotropes had other
functional receptors for different hormones in the peripheral
tissues in this early gestational age [14, 15]. In the in vitro
studies GH releases in the fetal tissues insulin-like growth
factors (IGF-I, IGF-II), increases the proliferation of pancreatic
beta cells, controls the synthesis of myelin in the brain and
influences the maturation of peripheral organs in the fetus
[15]. The role of ghrelin in the fetal period is not fully appreciated despite the fact that receptors for this peptide are
detected in numerous organs [8, 16]. Their role is mostly
involved in the maturation of organs [3, 4]. Ghrelin plays an
important role in pancreatic islets, being more abundant in
fetal pancreas compared to a mature organism [17]. The Date
et al. studies suggest that ghrelin might be a physiologic
regulator for the release of insulin from the beta cells [18].
Our knowledge about the release of ghrelin from fetal pituitary
somatotropes is severely limited. Whether ghrelin produced
in the fetal tract might stimulate the release of GH in somatotropes is still unclear. Is ghrelin detected in somatotropes
in our current work the same as the one found in alimentary
tract? Currently there are no answers to these questions.
Ghrelin particles with different molecular weights exert
Immunohistochemical assessment of ghrelin, GHS-R, GH, GHRH and somatostatin in pituitary glands of human fetuses
different physiologic actions [3,5,19]. Short chain of ghrelin
devoid of 4-5 amino acids, but having octanyl bonds are
functional and activate GHS-R1a
but to a lesser extent
compared to 28 amino acid particle [8, 20]. Given
and
, even in high doses, they do not release GH in newborn
mice and do not displace radiolabelled ghrelin from binding
sites in the somatotropes membranes. The antibodies used by
us do not differentiate different forms of ghrelin. Rubinfeld et
al. examined in vitro the influence of new ghrelin analogs on
the secretion of GH from fetal pituitary and showed that ghrelin analog is a potent stimulator for release of ghrelin from
fetal pituitary and that it increases the affinity to GHS-R [21].
The authors of described ghrelin analogs do not specify the
structure of the particle, but stress that its action is stronger
compared to human ghrelin as far as the release of GH from
the fetal pituitary. It also has higher affinity to the specific
receptor. Ghrelin detected by us in somatotropes is synthesized by these cells. Our assumptions are confirmed RT-PCR
studies that detect the ghrelin mRNA, so this protein is surely
produced by these cells and it might influence the GH release
from fetal pituitary in auto- and/or paracrine way. Rubinek et
al. point to a fact that an important role in this period of life in
the regulation of GH secretion is played by intracellular contact via adhesion molecules like N-cadherin and neural
adhesion molecules (N-CAP) [22]. Authors conducted studies
on fetal pituitary glands from 19-23rd week gestation and
showed that stimulation of N-CAP increases the GH secretion
by 40% to 60%, while damage to N-cadherin resulted in 40%
decreased secretion of GH. The growth hormone apart for
pituitary gland is also produced in the placental syncytiotrophoblast. Similar situation exists in case of ghrelin, but its
is detected in cytotrophoblast mostly in I trimester [23, 24].
The placental growth hormone does not cross the placenta to
the fetal circulation, so the GH present in the fetal blood is
mostly of fetal origin.
The GH release by fetal pituitary somatotropes is not pulsatile, and the presence of ghrelin and GHS-R in somatotropes
might indicate its auto- and paracrine action of ghrelin in the
release of GH. The expression of this receptor protein is very
high. The hypothalamic-pituitary axis in the fetal period seems
to be inactive, since in 9 out of 10 examined pituitary glands
we have found no GHRH expression and in all of them there
was lack of somatostatin. The observed expression of GHRH
in the 32 week old newborn that survived 4 days, might suggest that in this newborn the activity of the hypothalamicpituitary axis was just beginning. Rubinfeld et al. studied the
influence of cortistatin, a neuropeptide with high homology
with somatostatin, and showed that it suppresses the GH in
the culture medium of fetal pituitary [21]. It is possible that
during fetal life there are mechanism that block the hypothalamic-pituitary axis.
in vitro
in vitro
in vivo
Conclusion
Our studies show that fetal pituitary gland somatotropes
between 18-37 week of gestation express GH, ghrelin and
47
GHS-R. These data suggest that the release of GH in fetal
pituitaries might be mediated by ghrelin based on auto- or
paracrine action. In the fetal life the hypothalamic pituitary
axis is inactive and the process of its activation most probably
begins in the first week of the extrauterine life.
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J
Andrzej Kędzia
Sławomira 16, 61-063 Poznań, Poland
e-mail: [email protected]