Molecular Human Reproduction, Vol. 9, No. 8, 481-490,
August 2003
© 2003 European Society of Human Reproduction and Embryology
Article |
Expression of calcitonin gene-related peptide receptor components, calcitonin receptor-like receptor and receptor activity modifying protein 1, in the rat placenta during pregnancy and their cellular localization
Submitted on February 3, 2003; accepted on April 22, 2003
Department of Obstetrics and Gynecology, 301 University Blvd, Medical Research Building, Rm. 11.138, The University of Texas Medical Branch, Galveston, TX 77555-1062, USA
1 To whom correspondence should be addressed. e-mail: chyallam{at}utmb.edu
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Calcitonin gene-related peptide (CGRP), one of the most potent endogenous vasodilators known, has been implicated in vascular adaptations and placental function during pregnancy. The present study was aimed to investigate mRNA expression of CGRP-A receptor components, calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein 1 (RAMP1) in the rat placenta. Immunohistochemical staining of rat placentas obtained on day 18 of pregnancy using polyclonal anti-CRLR and RAMP1 antibodies revealed that labelling was specifically concentrated in the cytotrophoblast and syncytium in labyrinth, trophoblastic giant cells and basophilic cells in trophospongial cell layer, and endothelium and smooth muscle cells in fetal vessels. The intensity of staining was reduced in all these cells except in the syncytium in placentas obtained during labour. RT–PCR analysis showed that mRNA expression of CRLR and RAMP1 was significantly higher in the rat placenta from gestation day 17 to day 22, than during labour. During pregnancy, 17ß-estradiol inhibits, while progesterone stimulates, placental mRNA and proteins for CRLR and RAMP1. Antiestrogen, ICI 182780, increased, whereas antiprogesterone, RU 486, inhibited the expression of both CRLR and RAMP1. In summary, we demonstrate the presence and cellular localization of CRLR and RAMP1 in the rat placenta. Elevated placental CRLR and RAMP1 may be involved in CGRP-related increases in blood flow and therefore fetal growth and decreases at term labour may help minimize the blood loss.
Key words: calcitonin gene-related peptide/calcitonin receptor-like receptor/rat placenta/receptor activity modifying protein 1/steroids
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Introduction |
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Calcitonin gene-related peptide (CGRP) is a 37 amino acid neuropeptide and one of the most potent endogenous vasodilators known (Amara et al., 1982). CGRP is predominantly synthesized in the dorsal root ganglia (DRG), and primary afferent neurons extend CGRP-containing nerves to peripheral sites such as blood vessels (Sigrist et al., 1986). CGRP is present in the circulation, and plasma CGRP concentration increases during pregnancy in both humans and rats (Stevenson et al., 1986; Gangula et al., 2000). CGRP relaxes peripheral blood vessels by binding to its receptors, which are widely distributed in cardiovascular tissues (Wimalawansa, 1996). Potent vasodilatory effects of CGRP have been reported in the uterine artery of sheep and humans. The effects in women are significantly greater during pregnancy (Yang et al., 1992; Nelson et al., 1993). It has been reported that CGRP relaxes human chorionic plate vasculature (Firth and Pipkin, 1989), and CGRP dose-dependently reduces placental vascular resistance in an in-vitro perfused placental cotyledons (Mandsager et al., 1994), suggesting a beneficial role for CGRP in utero-placental vascular regulation. More recently, we have demonstrated that subcutaneous infusion of rats with CGRP8–37, an antagonist of CGRP, causes a significant dose-dependent reduction in pup weight with an increase in mortality rate (Gangula et al., 2002). Therefore, we suggested that endogenous CGRP plays a role in maintaining normal feto-placental development and fetal survival.
It is well recognized that calcitonin receptor-like receptor (CRLR) can function as either a CGRP receptor or an adrenomedullin receptor depending on the expression of the type of receptor activity modifying protein (RAMP) (McLatchie et al., 1998). RAMP1 presents the CRLR at the cell surface as a CGRP receptor, whereas RAMP2 and RAMP3 transport CRLR as an adrenomedullin receptor. Recently reported data suggest the presence of two types of CGRP receptors; CGRP-A and CGRP-B receptors (Chauhan et al., 2003; Yallampalli et al., 2002). We have previously demonstrated the presence and regulation of CGRP-B receptors in the rat placenta (Dong et al., 2002). However, the expression of CGRP-A receptor components, CRLR and RAMP1, in the rat placenta, and their regulation during pregnancy remain unknown. Therefore, the objectives of this study were (i) to examine the cellular localization of CRLR and RAMP1 proteins in the rat placentas, (ii) to determine the mRNA expression of CRLR and RAMP1 in the rat placentas and their regulation, and (iii) to measure changes in the [125I]CGRP binding sites in the placenta during pregnancy and labour.
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Materials and methods |
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Animals and treatments
All procedures were approved by the Animal Care and Use Committee of the University of Texas Medical Branch and complied with National Institutes of Health guidelines. Timed pregnant Sprague–Dawley rats were killed in a CO2 inhalation chamber on days 17, 18, 19, 20, 21 and 22 of pregnancy before and during labour (six animals for each gestational age). The placentas were removed immediately, cleaned of fat, fetuses and fetal membranes, and rinsed thoroughly in cold PBS solution.
To assess the regulation of placental CRLR and RAMP1 expression by steroid hormones, the rats were treated with different treatment regimens. 17ß-Estradiol (E2, 5 µg/rat) was given on day 17 to mimic the increases that occur naturally on day 22 of gestation. Progesterone (4 mg/rat/day) was given from day 20 to day 22 of gestation to maintain adequate progesterone levels, because circulatory progesterone concentrations declined from day 20 of pregnancy. As levels of both E2 and progesterone are steady during days 15–19, antiprogesterone, RU 486 (10 mg/rat/day) and antiestrogen, ICI 182780 (0.3 µg/rat) were given on day 17 to oppose endogenous steroid hormones and to evaluate their requirement for maintaining the receptor levels in the placenta. Placentas were obtained and frozen in liquid nitrogen and then stored at –70°C for further RNA isolation.
Immunofluorescent localization of CRLR and RAMP1
Placentas obtained from rats on day 18 of gestation, during labour, and treated with E2 or progesterone, were rinsed thoroughly in cold phosphate-buffered saline (PBS) (0.1 mol/l, pH 7.4) and fixed in Bouin fixative as described previously (Dong et al., 1999). After routine tissue processing procedures of dehydration in ascending grades of ethanol, cleaning in xylene, and infiltration with paraffin, the tissues were embedded in paraffin. Sections (5 µmol/l thick) were rinsed with 3% normal goat serum with Triton X-100 for 10 min at room temperature, and then incubated with avidin–biotin blocking buffer to reduce non-specific staining. The primary polyclonal antibody for CRLR/RAMP1 in 1% normal goat serum was applied to the slide and incubated overnight in a cold room (4°C). After washing with PBS on a shaker, the slides were incubated with fluorescein-conjugated secondary antibody, Alexa Fluor 594 (1:200, red; Molecular Probes, Inc., USA) at room temperature for 4 h. The slides were rinsed with PBS for 30 min and then mounted using 4,6-diamidino-2-phenylindole (Vector Labouratories, Inc., USA). The segments were covered with coverslips and viewed with an Olympus microscope with Image-ProPlus software (Olympus Optical Co., Ltd, Japan).
RT–PCR
Total RNA was extracted from the rat placentas by a single-step guanidine thiocyanate method (Chomczynski and Sacchi, 1987) using Trizol reagent (Gibco BRL, USA). First-strand cDNA synthesis was primed with oligo-dT (using 2 µg of total extranuclear RNA with 10 IU of reverse transcriptase and oligo-dT 12–18 as primer) at 42°C for 40 min, as described previously (Dong et al., 1997). Ten per cent of the resulting cDNA was used for amplification by PCR with 35 cycles. PCR primers were derived from the published sequences of CRLR and RAMP1 (Njuki et al., 1993; McLatchie et al., 1998). The primers used for amplification of the housekeeping gene, ß-actin, were derived from the rat ß-actin cDNA sequence (Nudel et al., 1983). The primer sequences were: CRLR: 5'-TGCTCTGTGAAGGCATTTAC-3' and 5'-CAGAATTGCTTGAA CCTCTC-3'; RAMP1: 5'-GAGACGCTGTGGTGTGACTG-3', and 5'-TCGG CTACTCTGGACTCCTG-3'; and ß-actin: 5'-GTCGACAACGGCTCCGG CA-3' and 5'-GTCAGGTCCCGGCCAGCCA-3'. PCR products, with the use of these primers, were identical (data not shown) to those of published sequences confirmed by direct double-strand sequencing (DNA Sequencing System, Promega Corp., USA). The relative concentrations of CRLR and RAMP1 mRNA were determined by densitometric analysis of the ethidium bromide-stained reaction products using Sigma Gel analysis system. The results are expressed as the ratio of the densitometric readings for CRLR or RAMP1 to ß-actin mRNA from the same tissue.
Radiolabelled CGRP binding assay
Membranes were prepared from rat placentas and radiolabelled CGRP binding assay was performed as previously described (Dong et al., 1998). Briefly, rat placentas were homogenized in ice-cold Tris–HCl buffer (50 mmol/l, pH 7.4) containing 0.32 mol/l sucrose 1 mmol/l dithiothreitol, 5 mmol/l EDTA, and 200 kIU/l of aprotinin. After centrifuging at 1000 g for 10 min at 4°C, the supernatants were further centrifuged at 10 000 g for 20 min at 4°C. The crude pellets were then resuspended in Tris–HCl buffer and centrifuged again. The final pellets were resuspended in Tris–HCl buffer, and the protein concentrations were determined and adjusted to 1 mg of protein/ml with assay buffer (Tris–HCl 50 mmol/l, KCl 10 mmol/l, aprotinin 100 kIU/ml, sodium azide 3 mmol/l). Membrane preparations were incubated with 10–11 mol/l [125I]CGRP with or without varying concentrations of unlabelled CGRP (10–14 to 10–7 mol/l) in a total volume of 300 µl assay buffer with 0.5% heat-inactivated BSA for 150 min at 4°C. After incubation, 600 µl of assay buffer was added to each tube and centrifuged at 12 000 g for 5 min at 4°C. The bound radioactivity remaining in the pellets was counted in a gamma counter. Specific binding was calculated by subtracting the labelled CGRP bound in the presence of 0.5 µmol/l unlabelled CGRP from the total amount of labelled CGRP bound, and the receptor density in the rat placentas was expressed as CGRP fmol bound per milligram of protein.
Statistics
Results are expressed as mean ± SEM. Data were analysed for statistical difference using the Student’s t-test or analysis of variance followed by Bonferroni t-test. Differences were considered significant at P < 0.05.
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Localization of CRLR and RAMP1 in rat placentas
Using immunofluorescent methods, we found that immunoreactive CRLR and RAMP1 were primarily localized in the labyrinth, trophospongial cell layer, and blood vessels in the fetal surface of the placenta (Figure 1 and Figure 2). Compared with rat placentas obtained on day 18 of gestation (A1, A2 and A3 in both figures), the placental sections from rats during labour showed reduced intensity of staining for both CRLR and RAMP1 in the trophospongial cell layer and fetal vessels (B1, B2 and B3 in Figures 1 and 2). Reduced staining for both CRLR and RAMP1 also occurred in the labyrinth, except in the syncytium. Control sections without primary antibody showed no specific staining in the segments from the rats on day 18 of gestation (Figure 1C) and during labour (Figure 2C).
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With higher magnification of the sections, the positive staining for CRLR/RAMP1 in the placenta at day 18 of gestation is visualized as red precipitate in the cytotrophoblast and syncytium in the labyrinth, trophoblastic giant cells and basophilic cells in the trophospongial cell layer, as well as in the endothelium and smooth muscle cells in the fetal vessels (Figure 3: A1–A4). Treatment with 17ß-estradiol (on day 17 of gestation, 5 µg/rat) resulted in reduced intensity of the staining in these cells with the exception of syncytium in which the staining remained the same (Figure 3). The positive staining for CRLR/RAMP1 in placenta from the rat during labour (Figure 4: A1–A4) displayed a similar pattern with the placenta treated with 17ß-estradiol. However, labour induced decreases in the expression of CRLR/RAMP1 in the cytotrophoblasts, trophoblastic giant cell, basophilic cells, endothelium, and smooth muscle cells were reversed by the treatment with progesterone (day 20–22 of gestation, 4 mg/rat/day) (Figure 4).
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Changes in CRLR and RAMP1 mRNA in rat placenta
RT–PCR analysis showed that mRNA both for CRLR (497 bp) and RAMP1 (250 bp) were expressed in rat placentas (Figure 5A and B). Densitometric analysis of these mRNA bands showed that mRNA expression of CRLR and RAMP1 in rat placentas were increased from day 17 to day 22 of gestation, and then decreased during labour, suggesting a gestational-dependent regulation of CGRP receptor components in rat placenta.
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Regulation of CRLR and RAMP1 mRNA by estrogens
We investigated whether E2 or ICI 182780, an antiestrogen, modulate placental CRLR and RAMP1 mRNA expression in pregnant rats. As shown in Figure 6A, a significant decrease (59.0 ± 3.0% versus 100% in control, P < 0.01) in CRLR expression in rat placentas was noted 48 h after E2 administration. On the contrary, a profound increase in placental CRLR was observed (141.3 ± 12.5% versus 100% in control, P < 0.01) 48 h after ICI 182780 treatment, implying that E2 inhibited placental CRLR mRNA expression. Similar effects of E2 on RAMP1 mRNA expression were observed in the rat placentas. As shown in Figure 6B, E2 significantly inhibited RAMP1 expression in rat placenta (68.5 ± 1.0% versus 100% in control, P < 0.01) and ICI 182780 treatment stimulated RAMP1 expression in the rat placenta (119.0 ± 3.4% versus 100% in control, P < 0.01), suggesting that E2 inhibited CGRP receptor components, CRLR and RAMP1.
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Regulation of CRLR and RAMP1 mRNA by progesterone
We examined whether progesterone or RU 486, an antiprogesterone, modulate placental CRLR and RAMP1 mRNA expression in pregnant rats. As shown in Figure 7A, progesterone given during days 20–22 of gestation attenuated the fall in placental CRLR at term labour (74.3 ± 2.8% versus 45.8 ± 0.9% in term labour, P < 0.01). RU 486 given on day 17 induced preterm labour (data is not shown) in all treated rats and significantly reduced placental CRLR mRNA expression (47.5 ± 8.5% versus 100% in control, P < 0.01). In addition, progesterone treatment attenuated the fall in placental RAMP1 at term labour (76.0 ± 6.9% versus 61.3 ± 2.5% in control, P < 0.05) (Figure 7B). RU 486 treatment significantly decreased placental RAMP1 mRNA expression (44.5 ± 5.5% versus 100% in control, P < 0.01). These results indicated that progesterone administration to rats during late pregnancy prevented the decline in placental mRNA for CRLR and RAMP1 seen during term labour, whereas RU 486 inhibited the expression of both these genes during pregnancy.
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Radiolabelled CGRP binding
As shown in Figure 8, specific high affinity [125I]CGRP binding sites were present in rat placentas on day 18 of gestation, and the binding sites declined substantially during labour (1265.00 ± 43.8 fmol/mg protein in day 18 versus 808.30 ± 139.10 fmol/mg of protein during labour, P < 0.05), implying that the CGRP binding sites in the rat placenta were decreased during labour.
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Discussion |
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We provide evidence in this study that CGRP-A receptor components, CRLR and RAMP1, are expressed in rat placenta. Primary distribution of CRLR and RAMP1 is in the cytotrophoblast and syncytium in the labyrinth, trophoblstic giant cells and basophilic cells in the trophospongial cell layer, as well as in the endothelium and smooth muscle cells of fetal vessels. The intensity of staining for both CRLR and RAMP1 in these cells was reduced except in the syncytium in placentas obtained during labour. The levels of mRNA for both CRLR and RAMP1 were higher in rat placentas obtained from day 17 to day 22 of gestation, than during labour. Estradiol-17ß administration to day 18 pregnant rats inhibited placental mRNA and protein expression both for CRLR and RAMP1, while ICI 182780 caused an increase in mRNA for both. Progesterone administration to pregnant rats during late gestation prevented the decline in placental mRNA and protein for CRLR and RAMP1 seen during term labour, whereas RU 486 inhibited the expression of both of these genes. Specific high affinity [125I]CGRP binding sites are present in placentas on day 18 of pregnancy and the binding sites declined during labour. Therefore, increases in placental CRLR and RAMP1 and CGRP binding sites during the fetal growth phase could play a role in reduced vascular resistance and increased blood flow through the placenta to maintain rapid fetal growth during late gestation. At term, decreases in these receptors could play a role in minimizing blood flow through the placenta and thus reduce blood loss during labour and delivery.
Circulating immunoreactive CGRP in adults has been suggested to come from the release of CGRP from sensory nerve endings, but the source of CGRP in the feto-placental circulation is not clear. Reports have shown that CGRP concentrations in cord blood are higher than those in the mother’s plasma at term (Parida et al., 1998), and therefore, it is possible that CGRP in the fetal circulation is derived from sensory neurons of the fetus, or even from the placenta itself. Immunoreactive CGRP in serum was first detected in the fetal rats at gestational day 18 (Gon et al., 1990) and then considerably increased between postnatal day 5 and day 14. This direct correlation of circulating CGRP in the fetal rat and newborn with increased mRNA and protein expression for the CRLR and RAMP1 in the placenta at late pregnancy reported in the current study implicate a role for the CGRP system in placenta in the development of the fetus. This is also supported by our previous in-vivo studies demonstrating a substantial decrease in both fetal and placental growth in rats when CGRP8–37, an antagonist of CGRP, was continuously infused from day 18 of gestation (Gangula et al., 2002).
The present study demonstrated for the first time that CGRP-A receptor components, CRLR and RAMP1, are localized in the blood vessels of the fetal surface and in the trophoblastic giant cells, cytotrophoblast, syncytium, and basophilic cells, implicating the involvement of CGRP in the control of placental blood flow and perhaps production of placental hormones.
The definitive placenta in the rat is formed on days 12–13 of gestation with two major regions, the labyrinth and the trophospongial cell layer (Rugh, 1968). The labyrinth region represents the major portion of the placenta and is composed of both maternal blood channels and fetal vessels. The maternal sinusoids of the labyrinth, however, were denuded of endothelium and instead were lined with cytotrophoblast, which is in direct contact with the maternal blood space, and the syncytium that lies just outside the core of vascular fetal mesenchyme (Enders, 1965). During later stages of pregnancy, this labyrinth barrier, with its intricate vascularization, is the primary route of maternal–fetal transport (Metz et al., 1978). The trophospongial cell layer is positioned between the labyrinth and the maternal decidua (Davies and Glasser, 1968). This placental zone is composed initially of undifferentiated trophoblastic giant cells that then differentiate into three different cell types. These consist of trophoblastic giant cells, basophilic cells, and glycogen cells (Davies, 1968). Glycogen is present in the glycogen cells, and basophilic cells can further differentiate to become trophoblastic giant cells. This area is vascularized only by maternal blood passing to and from the labyrinth and is the site of production and secretion of placental steroidal and luteotrophic hormones (Chan and Leathem, 1975). The present study demonstrated the existence of CRLR and RAMP1 in the fetal vessels, labyrinth, and trophospongial cell layer and the decreases of their intensity in the placenta during labour, implicating a role for CGRP in the control of vascular tone and perhaps the production of placental hormones in the feto-placental circulation.
We also noted that the intensity of the staining for both CRLR and RAMP1 were retained in the syncytium in the labyrinth of the placenta during labour. The significance and underlying mechanisms of this retention of CGRP receptors warrant further investigation. Although the major source of steroid hormones during pregnancy in the rat appears to be the corpus luteum, placental progesterone synthesis has also been described (Matt and MacDonald, 1985). Since the trophoblast cells are the site of steroid hormone synthesis (Jollie, 1981) in the placenta, the existence of CRLR and RAMP1 in the cytotrophoblast and syncytium in the rat placentas may imply that CGRP plays a role in steroid hormone synthesis by acting through its receptors.
In the present study, we demonstrated that progesterone given to rats during days 20–22 of pregnancy attenuates the fall in placental CRLR and RAMP1 at term labour. Further, RU 486 given on day 17 of pregnancy significantly inhibited placental CRLR/RAMP1 mRNA expression, suggesting that progesterone may be required for maintaining placental CGRP actions. We also demonstrated that the CRLR/RAMP1 in the rat placenta are substantially lower after E2 administration, indicating that E2 inhibits placental CGRP-A receptor expression. This concept is further supported by the ICI 182780-induced increase in placental CRLR/RAMP1 expression. It is apparent from this study that both E2 and progesterone regulate CGRP-A receptors in the rat placenta. A fall in progesterone during late gestation with a substantial surge in E2 at term may have a profound inhibitory effect on CGRP actions in placentas during labour.
Until recently, the identity and mode of action of receptors that bind CGRP have been uncertain. Several putative receptors in a variety of cells and tissues have been reported to bind CGRP and activate CGRP-induced secondary messengers. Our previous study has demonstrated that a monoclonal antibody (Wimalawansa and El-Kholy, 1993; Morara et al., 1998) that was raised against ligand affinity-purified CGRP receptor protein from porcine cerebellum does not react with CRLR when it is transiently expressed in human embryonic kidney cells either alone or in combination with RAMP1 (Yallampalli et al., 2002). Furthermore, Western blot analysis using this monoclonal antibody to cerebellar CGRP receptor protein demonstrated that CGRP receptor is expressed in the cerebellum, uterus, lungs and mesenteric artery of rats and in human myometrium, but not in SK-N-MC cells, a cell line that has been demonstrated to express CRLR and RAMP (McLatchie et al., 1998). In contrast, SK-N-MC cells and rat uterus, but not rat cerebellum, express mRNA for CRLR and RAMP1, further supporting the existence of two separate CGRP receptors: one CRLR-related CGRP-A receptor and the other unrelated to CRLR, CGRP-B receptor (McLatchie et al., 1998). Previously, we have demonstrated the expression of CGRP-B receptors in rat placenta and its regulation by steroid hormones (Dong et al., 2002). The present study extends our previous studies and provides evidence that, in addition to CGRP-B receptor, CGRP-A receptors are also present in the rat placenta and that similar to CGRP-B, levels of CGRP-A receptor components in placenta are increased with advancing gestation and decreased at term labour. Progesterone stimulated and estrogen inhibited placental CGRP-A receptor expression. Thus, the elevation in both CGRP-A receptors and B-receptors in the placenta during advanced stages of pregnancy could play a role in increasing blood flow through the feto-placental unit, and modulating placental secretory functions to support rapid fetal growth. A decline in CGRP receptors in the placenta at term labour (and upon RU 486 treatment) could reduce placental blood flow and therefore minimize blood loss during labour and delivery.
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Acknowledgements |
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This research was supported by the National Institutes of Health grants HD38324 (Dong), HD30273 (Yallampalli) and HL58144 (Yallampalli). We thank Ms Kimberly Mitchell for her excellent typing.
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