Molecular Human Reproduction, Vol. 7, No. 4, 379-385,
April 2001
© 2001 European Society of Human Reproduction and Embryology
Expression of galanin in human placenta
Department of Gynaecology and Obstetrics, University of Ulm, Germany
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Abstract |
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The neuropeptide galanin was originally implicated in the regulation of feeding behaviour. Today, galanin is implicated in several physiological functions including reproduction and feeding. Many hypothalamic neurohormones of the hypothalamo–pituitary axis (HPA) are also expressed in the placenta where the specialized topological compartments of the HPA are missing and where paracrine and autocrine regulatory mechanisms consequently prevail. Since galanin influences gonadotrophin-releasing hormone secretion in the HPA, we argued that a similar regulatory role for galanin might exist in human placenta. Since the presence of galanin in human placenta had not been previously reported, we analysed galanin expression in the human placenta by immunohistochemistry and quantitative polymerase chain reaction (PCR) throughout gestation. We found that the peptide hormone localizes to the syncytio- and cytotrophoblast layers; its RNA could be detected. By quantitative PCR we observed that throughout gestation, there is a loss of galanin mRNA which parallels the fall in signal intensity from immunohistochemical detection of the galanin oligopeptide. Furthermore, we detected secretion of galanin from isolated trophoblastic cells. We conclude that galanin may be an important and novel regulator of placental function.
galanin/gestation/immunohistochemistry/quantitative PCR/secretion
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Introduction |
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The neuropeptide galanin is expressed in the human brain, preferentially in the hypothalamus, and in areas of the pituitary (Ryan and Gundlach, 1996
; Gundlach and Burazin, 1998). Recent data (Wynick et al., 1998) in galanin gene-disrupted mice have established that galanin is indispensable for lactation, since in the defective mice the prolactin levels are drastically reduced and cannot be stimulated by oestradiol. The proliferative capacity of pituitary cells in response to oestradiol is also abolished. All the other reported functions of galanin in the neuroendocrine network [its role in feeding behaviour (Maiter et al., 1990); regulation of body weight (Leibowitz and Kim, 1992); modulation of gonadotrophin-releasing hormone (GnRH) or GnRH release (Lopez et al., 1991)] are not affected in these animals. Whether this reflects compensatory mechanisms in the knock-out mice or lack of involvement of galanin in other essential regulatory circuits, is not known at present. Nevertheless, the studies pointed to an important role of galanin in reproduction.
We have been interested in the co-regulation of galanin and reproductive neurohormones such as GnRH. In the hypothalamus, GnRH-producing neurons co-express two other hormones, delta sleep-inducing peptide and galanin (Charnay et al., 1989; Merchenthaler et al., 1990). Earlier studies in rats have shown that expression of galanin in GnRH neurons can be related to the onset of puberty (Rossmanith et al., 1994). The co-expression in individual cells is induced by oestradiol and facilitated by progesterone (Rossmanith et al., 1996). During the oestrus cycle, co-localization peaks in the oestrus when 85% of GnRH neurons simultaneously express galanin, and it falls again in di-oestrus (Marks et al., 1993b). This rise of galanin expression is only observed in GnRH neurons, not in neurons positive for galanin alone (Marks et al., 1993a). Furthermore, co-localization and co-expression is much stronger in normal female rats, and only marginal in males (Merchenthaler, 1998).
GnRH is one of the most important regulators of reproduction [for review see; (Lopez et al., 1998)]. In human chorion and placenta, GnRH induces chorionic gonadotropin secretion, which in turn is essential for rescuing progesterone production in the corpus luteum, thus enabling the developmental progression in early pregnancy. Given the co-expression of GnRH and galanin in the hypothalamus, we suspected a similar situation in placenta despite its lack of topological organization, and of neurons. Our initial aim was to investigate whether galanin was indeed expressed in the human placenta. In this paper we present data which demonstrate that galanin mRNA and protein is expressed in the human placenta and that the expression levels vary in early and late pregnancy.
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Materials and methods |
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Use of human tissue
Permission to use human material was granted by the local Ethical Committee prior to the study.
Preparation of tissue sections
Fresh placental tissue was paraffn-embedded after overnight fixation in formalin (Sigma, Deisenhofen, Germany). Serial sections (1µm) were mounted on slides, rinsed twice for 10 min in xylene, followed by rehydration in decreasing concentrations of ethanol.
Immunohistochemistry
Galanin was detected in a sandwich-antibody assay as previously described (Kleine et al., 2000) using rabbit anti-galanin serum (Chemicon, Hofheim, Germany; diluted 1:400 in phosphate-buffered saline: 10 mmol/l sodium phosphate, pH 7.2, 140 mmol/l sodium chloride) and the universal immunostaining kit (Immunotech, Hamburg, Germany). The presence of galanin was visualized by means of a stereo light microscope (Zeiss, Oberkochen, Germany). Using pre-immune rabbit sera (Kleine et al., 2000), we have previously established that with this dilution of rabbit sera only specific binding is detected by this method.
Preparation of isolated trophoblastic cells and culture of trophoblastic cells
Suspensions of human trophoblastic cells were prepared as published (Wolfahrt et al., 1998), using a protocol as previously described (Li et al., 1996). Following the mechanical and enzymatic dispersal of placental fragments, cells were washed in Ham's F-12/Dulbecco's modified Eagle's medium (DMEM; Sigma) and then separated according to their sizes by sedimentation into bovine serum albumin density gradient (1, 2 and 3% BSA, 1 h by gravity). The trophoblasts at the bottom of the tube were incubated with a mixture of monoclonal mouse antibodies against HLA-ABC (w6/32) and HLA-DR/DP/DQ (CR3-43; both from Dako). Afterwards they were washed and treated with sheep anti-mouse antibodies coupled to magnetic beads (Dynal, Hamburg, Germany). Cells positive for HLA antigens were retained in a magnetic field while HLA negative cells could be pipetted away. These latter cells were then used for further experiments. Purified cells were seeded into Petri dishes (Becton Dickinson, Heidelberg, Germany). Cells were cultured in Ham's F-12/Dulbecco's modified Eagle's medium DMEM (Sigma) containing insulin, selenit, EGF, dexamethasone (all from Sigma) at 37°C in a humidified atmosphere with 5% CO2 for 3 days.
Radioimmunoassay for galanin
The radioimmunoassay was performed according to the manufacturer's instructions (Peninsula Laboratories Inc., Belmont, CA, USA): supernatants from serum free trophoblastic cultures diluted 1:1 with radioimmunoassay buffer were incubated in parallel with a titration of human galanin standard provided by the manufacturer and a polyclonal rabbit anti-galanin antibody in 5 ml polystyrene tubes (Becton Dickinson) overnight. The 125I-labelled galanin tracer was added and incubation performed overnight again. Goat anti-rabbit antibodies and normal rabbit serum were then added, the samples were incubated for 90 min and centrifuged at 1700 g, the supernatant was removed and radioactivity in the precipitate was measured in a gamma counter (LKB Wallac). Using the standard curve and sigmoid regression
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Preparation of mRNA
Deep-frozen tissue samples together with solid carbon dioxide were placed into a mortar and ground into a fine powder. The powder was transferred into the trizol reagent (Life Technologies, Heidelberg, Germany). Debris was removed by centrifugation. The clear supernatant was extracted with chloroform, and the RNA was precipitated with isopropanol, washed and dried. After solubilization in water, aliquots were kept frozen at –80°CC. Additionally 50 µg RNA samples were treated with 50 units DNase I (Boehringer Mannheim), thereafter phenol/chloroform/isoamyl alcohol extracted, washed with 70% ethanol, dried and resolubilized in water. Quantification was performed by UV spectrometry (DU-600; Beckman Coulter, Fullerton, CA, USA).
Reverse transcription-polymerase chain reaction (RT-PCR)
mRNA was reverse-transcribed into cDNA using MMLV reverse transcriptase (Boehringer) and oligo-d(T)25 (Amersham Pharmacia Biotech, Freiburg, Germany) in RT buffer: 67 mmol/l Tris–HCl pH 8.8, 16.8 mmol/l (NH4)2SO4, 6.7 mmol/l MgCl2. The cDNA was amplified using primers listed in Table I. A volume of 50 µl consisting of reaction buffer (50 mmol/l KCl 10 mmol/l Tris–HCl, pH 8.3; Perkin Elmer, Norwalk, CT, USA), 1.5 mmol/l MgCl2, sense and antisense primers at 0.5 µmol/l concentrations, 200 µmol/l dNTP and 2 units of Taq polymerase (Perkin Elmer) was used. Cycling conditions were: pre-cycling: 3 min 94°C; cycling: 1 min 94°C, 1 min 59°C, 1 min 72°C; 35 cycles; post-cycling: 10 min 72°C.
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Preparation of a shortened galanin cDNA for quantitative estimation of galanin mRNA
Using the TA-cloning kit (Invitrogen, Groningen, Netherlands), a 400 bp fragment of the galanin cDNA was cloned into the pCR–2.1 vector. The insert of the pGal plasmid was sequenced using M13 primers and cycle sequencing. The identity of the inserted sequence to the published galanin sequence was established.
As shown in Figure 3, the pGal plasmid was digested with BbsI and SpeI (NEB, Beverly, MA, USA) removing a 311 bp insert and leaving a 4037 bp vector. Additionally, by HindIII (NEB) digestion of the pGal plasmid, a 205 bp fragment was cleaved off. The 4037 bp vector and the 205 bp fragment were each separated by agarose gel electrophoresis from other digestion products and isolated from the agarose using the gel extraction kit (Qiagen, Hilden, Germany). Sticky ends were then killed with Klenow Polymerase (NEB). After recutting with BamHI (NEB), both the vector and the 187 bp fragment were purified by phenol/chloroform extraction. The vector was further treated with alkaline phosphatase to remove terminal phosphates. Ligation was performed using T4 ligase (Invitrogen) with various ratios of vector to fragment. The new plasmid was transformed into one-shot bacteria (Invitrogen) and selected by blue/white screening. Of 24 colonies tested by PCR with the galanin primers, a product of the expected 270 bp length could be amplified from 21 colonies. The p
Gal plasmid was extracted from larger cultures using the Maxiprep Kit (Qiagen).
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Quantitative PCR
cDNA (resulting from reverse transcription of 5 µg total RNA) was amplified in the presence of graded doses of the p
Gal plasmid using the galanin and glyceraldehyde-6-phosphate dehydrogenase (GAPDH; EC 1.2.1.) sense and antisense primers (see Table I
). Products were separated on 2% agarose gels, photographed and electronically scanned, and evaluated using the Imagemaster 1D program (Amesham Pharmacia Biotec, Freiburg, Germany). The ratio of the intensities of the 400 bp band (sample) and of the 270 bp band (competitor) were plotted versus the competitor dose. The amount where both intensities were equal was read from the graph and the cDNA amount of the galanin calculated. cDNA galanin amounts from different placentae were normalized using the intensities of the respective GAPDH bands. |
Results |
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Immunohistochemical detection of the galanin oligopeptide in placental sections
Using a polyclonal antiserum against human galanin we established that galanin could be found in the human placenta. Figure 1
demonstrates that first and third trimester placenta stained positive for galanin. In the first trimester samples the trophoblastic layers were positive for galanin with occasional Hofbauer macrophages also staining positive. In the third trimester specimen, a more general staining pattern was observed (Figure 1), however, when multiple third trimester placentae were analysed, a less intense staining pattern was observed in more than five different term placentae.
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Detection and quantification of galanin mRNA
Using RT-PCR, we analysed galanin mRNA in placentae from different gestational ages. Figure 2
demonstrates that mRNA was found in all four samples from first trimester placentae (labelled 1st), but hardly visible in those from third trimester placentae (3rd). The MCF-7 cell line (a human breast carcinoma line used as positive control) was strongly positive. To address these qualitative differences in quantitative terms, we developed a quantitative PCR to measure amounts of galanin mRNA in relation to GAPDH mRNA, a well-known housekeeping gene in the human placenta (Dahia et al., 1997). The PCR product from Figure 2 was cloned and sequenced. From the isolated pGal plasmid we derived a shortened galanin sequence in the pGal plasmid using the strategy described in Figure 3.
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X174 HaeIII-digested; right pBR322 MspI-digested.The competitive multiplex PCR was set up with constant amounts of cDNA from different placentae of early and late gestational ages, varying amounts (amol doses) of the p
Gal plasmid and primer pairs for galanin and GAPDH. Figure 4 demonstrates one example, showing the three bands for the galanin sample (400 bp), the titrated pGal plasmid (270 bp; competitor) and the GAPDH fragment (206 bp). By image analysis we derived the ratio of sample/competitor and this was plotted versus the competitor doses. From these plots (compare Figure 5), we read the point where sample and competitor amounts were equal [log(sample/competitor) = 0; point of equivalence]. Finally we normalized the results from different figures by dividing through the GAPDH intensity. For nine first trimester placentae and nine placentae at term the GAPDH-normalized galanin cDNA estimates were plotted (Figure 6). Only five term placenta points are shown. The other four did not show a galanin band and were thus below the limit of detection. Overall levels of galanin mRNA were highest at the beginning of gestation and dropped to low and sometimes undetectable levels at the end of pregnancy.
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Secretion of galanin from isolated trophoblastic cells
We then sought to determine whether galanin is secreted by trophoblastic cells. For this purpose, we used cultures of isolated cytotrophoblastic and syncytiotrophoblastic cells as previously described (Wolfahrt et al., 1998
). The cells cultured were HLA negative and cytokeratin 18 positive (not shown). Pilot experiments had revealed that galanin was present in fetal bovine serum (not shown); therefore, we used serum-free cultures according to a previously published protocol (Li et al., 1996). By radioimmunoassay the galanin concentrations in supernatants of 24 h cultures from four different placentae were found to be in the picogram range (nanomolar concentrations) and one sample was below the limit of detection (Figure 7). Thus, galanin is indeed secreted by cells from the trophoblastic layers of human placenta. Secretion could be observed for longer culture periods when medium was changed daily showing that the galanin amounts in the supernatant are not merely leakage from dying cells (not shown).
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Discussion |
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This paper presents an analysis of galanin expression in human placenta. The experiments show the expression of the galanin gene, both qualitatively and quantitatively, the detection of the galanin oligopeptide and the fact of secretion of galanin by trophoblastic cells. This is to our knowledge the first confirmation of galanin expression in human placenta.
Earlier work (Graf et al., 1996) was unable to demonstrate the presence of galanin in term placentae. Our results expand on these originally negative findings by demonstrating that galanin expression is higher in the early placenta (first trimester), while the RNA levels at term are much lower, sometimes below the limit of detection. The quantitative RT-PCR was performed with a truncated variant of the galanin fragment amplified from placenta. We extended the competitive PCR to a multiplex PCR with an additional primer pair for GAPDH. By normalizing the galanin cDNA levels by GAPDH transcript intensities, we eliminated experimental variation during RNA preparation and RT. The results shown in this quantitative RT-PCR depend on the assumption that GAPDH is uniformly expressed during gestation. GAPDH has been used as standard in a variety of normal, neoplastic, or neuroendocrine tissues (Dahia et al., 1997), and the few reports of its use in placenta have not revealed a regulation during gestation (Freed et al., 1997; Yelich et al., 1997; Rossmanith et al., 1999).
The galanin oligopeptide was determined using a commercial anti-galanin rabbit antiserum or a sheep anti-galanin serum with similar results. The comparison also established a control for the commercial antiserum routinely used. At the dilution applied, rabbit pre-immune sera did not stain placental tissues as established earlier (Kleine et al., 2000). We determined that the galanin oligopeptide is present in both layers of placental trophoblasts and in Hofbauer cells. At present, we do not know the role of galanin in these cells. In the rat brain, galanin has been found to be co-expressed with GnRH (Merchenthaler et al., 1990). Since we have determined GnRH to be expressed in the trophoblastic layers and in Hofbauer cells (Wolfahrt et al., 1998), the galanin gene might be controlled by similar transcription factors as the GnRH gene. However, since GnRH expression remains constant during gestation (B.Kleine et al., unpublished data) while, as shown here, galanin gene expression declines later in pregnancy, transcriptional control might differ in early and late pregnancy. Whether galanin exhibits a specific function in human placental cells is not known. The galanin `knock-out' mice (Wynick et al., 1998) were fully viable when foster mothers were provided, showing that, at least in mice, galanin has no obvious role in placenta, or that its absence can be substituted for by other neuroendocrine hormones. In humans, mutations in the galanin gene which may be informative about function have not been reported, possibly because failure of lactation is a not uncommon phenomenon.
Another argument for a functional role of galanin may arise from the fact that galanin is indeed released from trophoblastic cells. Using a commercial radioimmunoassay, we determined galanin secretion from cultured human trophoblastic cells. According to the protocol applied and to previous controls, these cells were devoid of HLA antigens. By positive cytokeratin 18 staining we have also established (S.Wolfahrt et al., unpublished data) that the cells were villous and not extravillous trophoblasts. Thus, we ascertain that the cells seeded into culture were either villous cytotrophoblasts or syncytiotrophoblasts; we excluded HLA positive Hofbauer cells (occasionally found to be galanin positive) or extravillous trophoblasts [which are HLA-AB negative but HLA-C and -G positive; (Verma et al., 1997)]. All other cells in human placenta are HLA positive per se. Since we used the previously published serum-free cell culture system (Li et al., 1996), we omitted galanin contamination from fetal serum sources. Secretion of galanin during the first 24 h of culture was in the nanomolar range. Starting with about 1 or 2x106 cells per ml, the rate of cellular galanin secretion was about 1400 molecules per cell per second assuming that the rate of secretion was equal amongst all cells. When compared to the synthesis rate of highly specialized immunoglobulin-secreting plasma cells [2000 molecules per second per cell; (Kindt and Capra, 1984)] the magnitude of secretion is similar. The amount of galanin secreted, therefore, supports the hypothesis for a functional role even if we do not know at present what this role may be.
Our study also showed the presence of galanin in fetal bovine serum. While establishing an enzyme immunoassay with supernatants from cells cultured in the presence of fetal bovine serum, we detected galanin in the day 0 controls and also in the culture medium itself. The use of bovine calf sera for the study of placental neuropeptides is therefore not advisable.
Taken together, we have established by different techniques that galanin, until now known as an important part of the hypothalamic-pituitary axis, is also expressed, synthesized and secreted in human placenta where it plays a physiological role yet to be defined.
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Acknowledgements |
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We are indebted to Prof. Istvan Merchenthaler (Wyeth Ayers Research, Radnor, PA, USA) for his generous gift of the anti-galanin antibodies. Special thanks to Prof. Ashley Grossman for his critical comments in editing the manuscript. This work was supported by Bausteinfoerderung of the University of Ulm and by grants from the Deutsche Forschungsgemeinschaft (Bonn, Germany) to W.G.R. (Ro657/6-4).
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Notes |
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1 To whom correspondence should be addressed at: Zentrum fuer klinische Forschung, University of Ulm Helmholtzstr. 8-1, D-89081 Ulm, Germany. E-mail: bernhard.kleine{at}medizin.uni-ulm.de
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References |
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Submitted on September 25, 2000; accepted on December 27, 2000.
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