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Department of Medicine (O.G., F.F.C.), Molecular Endocrinology Section and Complejo Hospitalario Universitario de Santiago; Department of Physiology (J.E.C., C.D.) and Department of Morphological Sciences (M.B., T.G.-C.), University of Santiago de Compostela, School of Medicine, Santiago de Compostela, Spain; and Department of Biochemistry (M.K., K.K.), National Cardiovascular Center Research Institute, Osaka, Japan
Address all correspondence and requests for reprints to: Felipe F. Casanueva, Department of Medicine Molecular Endocrinology Division, University of Santiago de Compostela School of Medicine, S. Francisco S.N., 15705 Santiago de Compostela, Spain. E-mail: melage{at}usc.es
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Abstract |
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Introduction |
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On the other hand, the placenta is a short-lived organ able to express and release a variety of hormones and with a complex and badly understood regulation. Several members of the somatotrophic axis, such as GH, GHRH, IGF-1, somatostatin, and GH variants have been detected in placenta of both human and experimental animals. While some hormones are secreted in a statical form, i.e. without changes along pregnancy, others are time related. To verify if ghrelin tissue expression was present in a relevant endocrine tissue as placenta, the tissue distribution and pregnancy-related variations of ghrelin were assessed in both human and rat placenta.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TM
method as previously described (5) (Life Technologies, Inc., Grand Island, NY). Tissues (about 100 mg)
were homogenized using a Polytron homogenizer
in 1000 µl of Trizol LS reagent and, recovery of total RNA after
isopropanol precipitation, was measured with a spectrophotometer
(Beckman Inc., Fullterton, CA; DU62) at 260 nm.
RT-PCR and Southern analysis
Two micrograms of total RNA were used to perform RT-PCR.
Complementary DNAs were synthesized using 200 U of Moloney murine
leukemia reverse transcriptase (Life Technologies, Inc.)
and 6 µl of dNTPs mix (10 mM of each dNTP), 6 µl of
first strand buffer [250 mM Tris-HCl pH 8.3, 375
mM KCl, 15 mM MgCl2
(Life Technologies, Inc.)] 1.5 µl of 50 mM
MgCl2, 0.17 µl of random hexamers solution [3
µg/µl (Life Technologies, Inc.)], 0.25 µl of RNase
Out [recombinant ribonuclease inhibitor 40 U/µl (Life Technologies, Inc.)], in a total volume of 30 µl. Reaction
mixtures were incubated at 37 C for 50 min and at 42 C for 15 min. The
RT reaction was terminated by heating at 95 C for 5 min and
subsequently quick chilled on ice. Three microliters of RT reaction
were used for PCR amplification. The amplification conditions were as
follows: 5 µl of PCR buffer [200 mM Tris-HCl pH 8.4 and
500 mM KCl (Life Technologies, Inc.)], 1.5
µl of 50 mM MgCl2, 4 µl of dNTPs
mix, 150 ng of rat ghrelin upstream primer 5'-TTGAGCCCAGAGCACCAGAAA-3',
150 ng of rat ghrelin downstream primer 5'AGTTGCAGAGGAGGCAGAAGCT-3'
(from GenBank AB029433), or human ghrelin upstream primer
5'-TGAGCCCTGAACACCAGAGAG-3' and human ghrelin downstream primer
5'-AAAGCCAGATGAGCGCTTCTA-3' [from GenBank AB029434) and 1.25 U of
Taq DNA Polymerase (Life Technologies, Inc.)].
The amplification profile for rat ghrelin was: denaturation at 98 C for
10 sec, annealing at 55 C for 30 sec, and extension at 72 C for 1 min
while for human ghrelin was: denaturation at 98 C for 20 sec, annealing
at 58 C for 30 sec, and extension at 72 C for 1 min. Thirty five- cycle
amplification was completed with an additional step at 72 C for 10 min.
The amplification was performed in an automatic thermal cycler
(Mastercycler gradient-Eppendorf). To assure that PCR was
performed in the linear amplification range, samples were taken after
15, 20, 25, 30, 35, and 40 cycles, showing that the reaction was linear
over this range (data not shown).
To check the quality of mRNA in each sample, rat ghrelin RNA was amplified together with ß-actin. For ß-actin, two specific primers span introns were used, which do not coamplify processed pseudogenes (6): forward primer (5'-TACAACTCCTTGCAGCTCC-3') and reverse primer (5'-ATCTTCATGAGGTAGTCAGTC-3'). PCR generates a single 347-bp product for rat ghrelin and a single 603-bp product for the ß-actin gene.
Human ghrelin RNA was amplified together with hHPRT: forward primer (5'-AGCAAGACGTTCAGTCCTGTC-3') and reverse primer (5'-CAGCCCTGCCGTCGTGATTA-3'). PCR reaction generates a single 327-bp product for rat ghrelin and a single 139-bp product for the hHPRT.
Multiple positive and negative controls were performed for RT-PCR. Negative controls consisted of omitting the RT reaction for each sample or amplifying samples of RT reaction without MMLV. To exclude a competitive amplification of genomic DNA, we have performed RT-PCR on rat genomic DNA. All negative controls resulted in no bands after amplification. The identity of the amplimer for ghrelin was confirmed performing the RT-PCR together with positive controls (purified cloned cDNAs for rat and human ghrelin: free insert and whole plasmid encoding the peptide). PCR products were separated on 1.5% agarose gel, stained with ethidium bromide and examined with UV light and visualized with a Gel Doc 1000 Documentation System (Bio-Rad Laboratories, Inc., Hercules, CA). To confirm authenticity of the amplimers, Southern blot analysis was carried out. Hybridization of nylon transferred resolved amplicons was performed using a 32P cDNA specific antisense probe for rat ghrelin. Hybridization was carried on at 44 C for 18 h. After removing the excess of labeled probe by washing, membranes were exposed to autoradiography, and the sizes of the bands determined by comparison with molecular weight marker on the ethidium bromide stained agarose gel pictures.
Northern blot analysis
For these studies, placentae and other tissues were dissected
from female Sprague Dawley rats as above described and snap frozen on
dry ice or immediately processed. RNA was extracted by a guanidium
isothiocyanate-phenol method, and 25 µg of total RNA were
fractionated by agarose gel electrophoresis. The 28s and 18s RNA bands
were visualized by ethidium bromide staining with a Gel Doc 1000
Documentation System (Little Chalfont, Buckinghamshire, UK).
RNA was transferred by capillary blotting onto a charged nylon membrane and fixed with UV light. The filter was hybridized to a 32P-labeled cDNA probe (501 bp) to rat ghrelin. Rat ghrelin cDNA was purified from plasmid expression vector with Sephaglas BandPrep Kit (Amersham Pharmacia Biotech). The probe was prepared by random prime labeling the rat ghrelin cDNA subcloned in EcoRI double restriction site of a pBS-SK(-) expression vector. Hybridization was performed overnight at 42 C in prehybridization buffer contain the labeled probe (1 x 106 cpm/ml), 6 post hybridization washes were performed before to expose the filter to x-ray film. Multiple exposure times, followed by densitometric analysis, were performed to ensure that the relative signals obtained indicated actual changes in mRNA levels and not merely artifacts due to any nonlinearity in film exposure. Relative mRNA levels obtained at different exposure times gave similar quantitative alterations. A rat 18S ribosomal RNA oligonucleotide probe 5'-ACGGTATCTGATCGTCTTCGAACC-3' (Amersham Pharmacia Biotech) was used to assess the amount and integrity of total RNA loaded in each gel. The oligonucleotide was end-labeled with 32P by polynucleotide kinase (Promega Corp.) and hybridized with filters in a buffer containing 10x Denhardt’s solution, 0.1% SDS, 2x SSC, and 100 µg/ml salmon sperm DNA at 50C for 24 h. The filters were then successively washed and processed as previously described.
Immunohistochemistry
Specimens of rat stomach and placenta were immersion fixed in
4% buffered formaldehyde for 24 h, dehydrated, and embedded in
paraffin by standard procedure. Sections 5-µm thick were mounted on
silanized slices, dewaxed, rehydrated, and immunostained by the
streptavidin-biotin peroxidase complex (SABC) procedure. The sections
were consecutively incubated in the following: 1) Rabbit anti ghrelin
polyclonal antibody, at a dilution of 1:500 in PBS (0.01 M
phosphate buffer, pH 7.4, containing 0.15 M NaCl) with
0.1% BSA (Sigma, St Louis, MO), for 1 h at room
temperature. 2) 3% hydrogen peroxide for 10 min. 3) Biotinylated goat
antibody to rabbit immunoglobulins at 1:100 dilution in PBS with 2%
normal goat serum (Duet kit, Dakopatts, Glostrup, Denmark) for 30 min.
4) Streptavidin-biotin-peroxidase complex (Duet kits, Dakopatts),
prepared 30 min before use according to the protocol provided by
manufacturer. 5) 3,3'-diamino-benzidinetetrahydrochloride (DAB)
solution for 10 min. DAB solution was prepared by dissolving 1 DAB
buffer tablet (Merck, Darmstadt, Germany) in 10 ml of
distilled water. Between steps the sections were washed with PBS
(2 x 5 min) and after step 5 with distilled water. No
counterstaining was done. Negative controls were performed by either 1)
omitting any essential step of the reaction; 2) substituting the
ghrelin antibody with appropriate dilution of normal (nonimmune) mouse
serum (Dakopatts); and 3) preabsorbing the anti ghrelin antibody with
the homologous antigen (ghrelin 10 nmol/ml) for 24 h at 4C.
Animal models
To verify ghrelin expression in rat in physiological models,
ghrelin mRNA expression along pregnancy was analyzed by Northern blot
analysis in placenta and in stomach. Female pregnant Sprague Dawley
rats were euthanized at different times of pregnancy: 8, 12, 16, 19,
and 21 days. Placentae and stomach were dissected as above described
and snap frozen on dry ice. Samples were immediately used for Northern
analysis or stored at -80 C until use. Stomach RNA from post coitus
female rats was used as reference in the study performed on rat
stomach.
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Results |
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, ghrelin mRNA was found in human
postpartum normal placenta samples by using RT-PCR analysis. This
result was also confirmed performing RT-PCR on human placentae at first
trimester (data not shown). The identity of the amplimers was confirmed
by restriction enzyme cleavage of the amplicons with SacI,
which originated two products: one of 75-bp and another of 252 bp,
positive control of restriction digestion was performed on the
amplification product of the purified cloned human pre-pro ghrelin cDNA
(data not shown). Interestingly, and as a complementary result, ghrelin
was also detected in an human tumor cell line as coriocarcinoma BeWo
cells (Fig. 1), although this expression was quite lower than in normal
human placenta.
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) with a peculiar distribution
pattern. In fact, ghrelin was mainly expressed in cytotrophoblast cells
and to a lesser extent in syncytiotrophoblast cells. In human stomach,
used as positive control (Fig. 2C), ghrelin was found in the
neuroendocrine cells of the fundic glands. Interestingly, ghrelin
expression vanished with the evolution of pregnancy, and in full-term
human placentae after delivery peptide levels were so low that
immunostaining became negative (Fig. 2B). To detect if these findings
were species specific, we have performed RT-PCR analysis on rat
samples. This analysis revealed the presence of ghrelin transcripts in
21 days pregnant rat placenta (Fig. 3).
The identity of the amplimers was confirmed by restriction enzyme
cleavage of the PCR amplification products with ApaI, which
originated two products: one of 138 bp and another of 209 bp. Positive
control of restriction digestion was performed on the amplification
product of the purified cloned rat pre-pro ghrelin cDNA, furnishing
identical results (data not shown). To verify the authenticity of the
amplicons, we have performed Southern analysis of PCR amplification
products with a specific cDNA antisense probe for rat ghrelin. As shown
in Fig. 3, B and D, ghrelin was easily identified in rat placenta, as
well as in other tissues in which ghrelin was previously described
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and 4).
In the rat placenta, immunoreactivity for ghrelin was demonstrated in
labyrinth trophoblast cells (Fig. 5, A
and B, middle and right), whereas the spongiotrophoblast and
secondary giant trophoblasts of the junctional layer were negative
(Fig. 5A, left). In the gastric sections, used as positive
control, immunoreactivity for ghrelin was strongly observed (Fig. 5C)
in the cytoplasm of neuroendocrine cells primarily situated at the base
of fundic glands.
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(upper panel), a pattern of
expression coincident with that found in human placenta was observed in
rat placenta. No detectable signals for ghrelin were observed in
placenta at day 8 and 12 of pregnancy afterwards, a clear increase of
expression was observed at day 16 while at days 19 and 21 ghrelin
expression decreases up to lower levels. On the contrary, no
differences were observed in ghrelin mRNA expression in rat stomach.
The densitometric analysis of the bands, normalized by OD in arbitrary
units of 18S RNA, is reported in Fig. 7.
Moreover, Northern analysis of 16 days pregnant uteri did not show any
signal, as well as nonpregnant uteri. However, a clear band was
observed in 16 days placenta, as previously observed, confirming that
the result obtained is due only to placental ghrelin RNA and not to
decidual and/or other placental membranes contamination (Fig. 4C). In
addition, immunohistochemistry of 8 days pregnant uterus did not show
any immunoreactive signal for ghrelin (Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These results are suggesting that, with advanced differentiation of placental tissues, ghrelin vanishes. However, one of the most significant findings was the time of pregnancy-related changes in ghrelin expression observed in rat samples. These observations powerfully suggest that ghrelin is not just a marker of placental tissue, but that it undertakes a physiological role in placental function. Whether this role consists in placental regulation of peripheral functions or if it is needed for placental development is not possible to ascertain at present.
It is not possible to ascertain at present the role of placenta-derived ghrelin in either the pregnancy period or in the fetal physiology. One of the peculiar features of placental endocrinology is that in some instances it mimics the function of other endocrine glands. Interestingly, all the main regulatory components of the somatotrophic axis appear to be present in the placenta (7, 8, 9, 10). Available evidences demonstrate the presence of GH regulatory hormones that are prevalently hypothalamic. For instance, immunoreactive GHRH and its mRNA have been reported in human placenta (11), and GHRH mRNA was also localized to the cytrophoblast in mouse and rat placenta by in situ hybridization (12, 13). Somatostatin, the GH-inhibiting hormone, has been found in human placenta (14) by immunohistochemical studies revealing its localization in the cytotrophoblast and in the stroma of placental villi. Notably, its pattern of expression resembles the one reported here for ghrelin, decreasing at the end of gestation (15). Moreover, the new discovered hormone leptin was found in human placentae and its localization was consistently observed in the cytoplasm of syncytiotrophoblast cells (16). Finally, IGF-1 is expressed primarily in syncytiotrophoblast, whereas IGF-2 mRNA appears in placental cytotrophoblast localized in the superficial basal plate (17, 18). IGF-1 mRNA levels are highest at first trimester, slightly decreased by second trimester and lowest at term. IGF-2 reach a peak of expression in the second trimester, while lower levels are observed in the first trimester and normal term (19, 20). The presence of ghrelin in placenta here reported suggests its involvement in a model of fetalmaternal interaction throughout paracrine, autocrine as well as endocrine communication.
To date, several possible functional involvements of ghrelin could be assigned during intrauterine development, such as the local modulation of GH release and/or the influence on maternal and/or fetal pituitary GH secretion. The physiological relevance of this finding is yet unclear but several possibilities can be pointed forward (21). Placenta-derived ghrelin could influence fetal growth and maturation. In support of this, it has been shown that exogenous GH administration increase placental and fetal growth, whereas GH deficiency is associated with fetal growth retardation (22). Whether alteration in ghrelin- gene expression could lead to growth deficiency and/or intrauterine growth retardation needs to be established. Interestingly, the ghrelin receptor is closely located to the map position of the Brachmann-de-Lange Syndrome, a pre and postnatal growth deficiency (23). Finally, ghrelin could act by regulating the fetal hypothalamus-pituitary-adrenal axis, since it has been shown that GHS administration to adult rodents and human leads to increased circulating level of ACTH and cortisol (24), although whether this also happen in the fetal period is yet unknown. In any case, the finding that ghrelin in placenta shows a period of pregnancy-related expression, a fact not paralleled in gastric tissue, suggest how more than a tissue reporter, ghrelin may have physiological functions in gestation.
In conclusion, the stomach-derived somatotroph-controlling hormone ghrelin is expressed in both human and rat placental tissues, with a peculiar cellular pattern of distribution. Ghrelin expression rises at mid pregnancy decreasing thereafter, a fact suggesting a physiological regulatory role.
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Acknowledgments |
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Footnotes |
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2 Recipient of a TMR 30 Research Training Grant, Program IV Framework
of RTD: Contract ERBFMBI-CT-98-3368 from the European Commission, DG
XII Science Research & Development.
3 Recipient of a predoctoral fellowship from University of Santiago
de Compostela.
4 Recipient of a Student’s fellowship from the Diputación de A
Coruña.
Received August 9, 2000.
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) (A). Immunostaining of third trimester placenta was negative (B).
Immunoreactivity of human stomach is present in neuroendocrine cells
situated at the base of fundic glands (C). In the control (stomach)
performed to validate the specificity of the immunoreaction obtained by
preabsorbing the anti-ghrelin antibody with the homologous antigen
(ghrelin), no signal was observed (D).
