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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 7 3477-3485
Copyright © 2004 by The Endocrine Society

Increased Expression of the Relaxin Receptor (LGR7) in Human Endometrium during the Secretory Phase of the Menstrual Cycle

Courtney P. Bond, Laura J. Parry, Chrishan S. Samuel, Helen M. Gehring, Fiona L. Lederman, Peter A. W. Rogers and Roger J. Summers

Department of Pharmacology (C.P.B., R.J.S.), Monash University, Clayton, Victoria 3800, Australia; Department of Zoology (L.J.P., H.M.G.) and Howard Florey Institute of Experimental Physiology and Medicine (C.S.S.), University of Melbourne, Victoria 3010, Australia; and Centre for Women’s Health Research (F.L.L., P.A.W.R.), Department of Obstetrics and Gynaecology, Monash University, Clayton, Victoria 3168, Australia

Address all correspondence and requests for reprints to: Professor R. J. Summers, P.O. Box 13E, Monash University, Victoria, Australia 3800. E-mail: roger.summers{at}med.monash.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Relaxin (RLX) is a structural homolog of insulin that is the ligand for the LGR7 receptor. Although the 6k peptide is produced by the ovaries to cause connective tissue remodeling of the rodent and pig reproductive tracts to facilitate parturition, in human reproduction, the role of RLX is less well understood. Binding of human gene 2 (H2) [33P]-RLX, expression of RLX peptides and the LGR7 receptor was examined in the human uterus at different stages of the menstrual cycle. A significant increase in RLX receptor binding in endometrium was identified by quantitative autoradiography in the secretory compared with the proliferative phase. H2RLX competed with [33P]-H2RLX binding with higher affinity than porcine RLX during both the proliferative and secretory phases. Increased LGR7 receptor gene expression during the secretory phase paralleled the changes in [33P]-H2RLX binding. Human gene 1 RLX transcripts were not detected in the uterus, and H2RLX gene expression was low and not influenced by the stage of the menstrual cycle. The studies show that binding to and gene expression of the LGR7 RLX receptor changes markedly with the phases of the menstrual cycle, suggesting a specific role for the hormone in the physiology of the human uterus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HETERODIMERIC PEPTIDE hormone relaxin (RLX) is a structural homolog of insulin that activates its own distinct receptor, now known to be LGR7, a member of the leucine-rich repeat family of G protein-coupled orphan receptors (1). Evidence that these receptors are present in the human uterus includes the identification of an LGR7 transcript (2), the stimulation of cAMP production by RLX in cultured human endometrial cells (3), and the demonstration of binding sites for biotinylated porcine RLX (PRLX) in both the endometrium and myometrium with substantial binding in the luminal and glandular epithelium (4). Specific RLX binding has also been shown in marmoset endometrial stromal cells, with weak signals observed in the epithelium (5). Biotinylated RLX binding is highest in the endometrial stroma during the secretory phase of the cycle and in early pregnancy.

The presence of circulating RLX available to act on these receptors has also been demonstrated. Humans have three RLX genes, designated H1 (6), H2 (7), and H3 (8), of which recombinant human gene 2 RLX (H2RLX) is the predominantly expressed gene and circulating peptide. Plasma levels of RLX are measurable during the luteal phase of the menstrual cycle and increase 6–7 d after the LH surge (9). The source of H2RLX in cyclic women is the corpus luteum (10), with substantial gene expression observed in the late luteal phase of the menstrual cycle. However, it has been recently shown (11) that local synthesis of H2RLX occurs in human endometrial stromal and glandular epithelial cells taken from both proliferative and secretory phases of the menstrual cycle. Immunoreactive RLX has also been localized to decidualized stromal cells in the late secretory phase and early pregnancy (12), implying that the endometrium provides an additional source of RLX in women.

Several studies show that RLX has a broad range of effects on the human endometrium and may mediate stromal cell differentiation and/or vascularization. RLX stimulates production of a number of factors from endometrial cells in vitro including prolactin (13), IGF binding protein-1 (14), glycodelin (15), and vascular endothelial growth factor (VEGF) (11, 16). The effects of RLX on VEGF secretion differs, depending on the stage of the menstrual cycle, with a negative influence observed in the proliferative phase and a stimulatory effect in the secretory phase (11). Of particular interest is the ability of RLX to stimulate VEGF expression in the secretory phase of the cycle, and the suggestion that the increased incidence of menometrorrhagia in RLX-treated patients is due to increased VEGF production (16).

The recent identification of LGR7 as the RLX receptor (1) has facilitated comparative studies among gene expression, function, and receptor binding. In this study, we used human endometrial tissues obtained during the proliferative and secretory phases of the menstrual cycle to examine receptor binding and LGR7 gene expression. Using qualitative and quantitative autoradiography, H2RLX binding was identified in human endometrium, and specific RLX binding sites were demonstrated in the epithelium of the endometrial glands and uterine lumen. The density of these epithelial RLX binding sites was dramatically increased in the early secretory phase of the menstrual cycle and was paralleled by a similar increase in LGR7 mRNA. Another objective was to examine LGR7 expression in relation to endometrial RLX and VEGF expression because RLX is known to stimulate VEGF secretion in the secretory phase of the cycle. Our data confirm the existence of specific RLX receptors in the human endometrium and support the hypothesis that RLX may be an important mediator of endometrial differentiation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Human tissue collection and preparation

Full-thickness endometrial tissue was collected from 27 hysterectomy operations performed on ovulating women suffering from nonmalignant gynecological disorders such as menorrhagia, fibroids, or uterine prolapse (age range 34–46 yr). Subjects had not received exogenous hormones in the 3 months before hysterectomy, and endometrial tissue was included in the study only if it appeared normal by routine histological examination. Ethical approval for this study was obtained from Southern Health Human Research and Ethics Committee B, and informed consent was received from each subject. Tissue blocks containing the intact endometrial layer and several millimeters of myometrium were frozen in liquid nitrogen and stored at –80 C.

Frozen blocks were oriented so that sections would be cut at right angles to the endometrial luminal surface, sectioned in a cryostat at –18 C, and collected on precleaned microscope slides coated with poly-L-lysine. After routine hematoxylin and eosin staining, tissue samples were sorted into seven groups according to the phases of the menstrual cycle by an experienced histopathologist using established criteria for the normal menstrual cycle (17). The seven phases of the menstrual cycle were early proliferative (EP), midproliferative (MP), late proliferative (LP), early secretory (ES), midsecretory (MS), late secretory (LS), and menstrual (M). Frozen sections (10 µm) were then used for quantitative autoradiography to determine expression levels and location of the RLX receptor.

For RNA extraction, endometrium was removed from the myometrial layer before freezing the tissue. Total RNA was extracted from thin sections of the frozen endometrial specimens homogenized in TRIZOL reagent (Invitrogen Life Technologies, Mt. Waverley, Melbourne, Australia) according to the manufacturer’s protocol. Purity of the RNA was enhanced by precipitating the aqueous phase of the TRIZOL preparation with an equal volume of 100% ethanol, followed by a second extraction through an RNeasy column (Qiagen, Hilden, Germany). After resuspension in RNase-free water, RNA was precipitated overnight with ethanol as previously described (18).

Quantitative autoradiographic measurement of receptor expression and properties using [33P]-H2RLX

H2RLX (B33) was labeled with [{gamma}-33P]-AMP (ATP) using the catalytic subunit of cAMP-dependent protein kinase and purified according to Tan et al. (19).

Slide-mounted uterus sections were preincubated in 100 µl HEPES buffer [25 mM HEPES and 300 mM KCl (pH 7.2)] containing phenyl-methylsulfonylfluoride (PMSF, 1 µM) for 30 min in a humidity chamber (25 C). The HEPES buffer was removed and slides were incubated for 90 min with 100 µl HEPES buffer with BSA (1 mg/ml), containing 100 pM [33P]-H2RLX for receptor localization, 50–1500 pM [33P]-H2RLX for saturation binding, or 100 pM [33P]-H2RLX in the absence or presence of increasing concentrations of unlabeled H2RLX or PRLX (10 nM to 10 µM) for competition binding. All nonspecific binding was defined with H2RLX (1 µM).

Labeled sections were washed (2 x10 min) in HEPES buffer, briefly rinsed in distilled water, and dried with a stream of air. Sections were apposed to a PhosphorImager plates for 3 d and then scanned with PhosphorImager SI (Molecular Dynamics, Sunnyvale, CA). Images were analyzed with ImageQuaNT (version 4.1; Molecular Dynamics). Receptor expression and saturation binding images were expressed as arbitrary phosphorimager units that were converted into disintegrations per minute per square millimeter using [{gamma}-33P]-ATP standards made by mixing concentrations of [{gamma}-33P]-ATP with rat ventricle paste, molding into cylinders, frozen, cut in a cryostat (10 µm), and either mounted onto poly-L-lysine-coated microscopes slides to be apposed with slide-mounted uterus sections or placed in liquid scintillation vials for direct determination of radioactivity. Competition binding was expressed as a percentage of total specific binding.

Uterus block numbers were then organized according to menstrual phase and data grouped using PRISM (GraphPad Inc., San Diego, CA).

Qualitative autoradiography of the distribution of [33P]-H2RLX binding

Slide-mounted uterus sections were preincubated in 100 µl of HEPES buffer containing PMSF (1 µM) for 30 min. Slides were then incubated with 100 µl HEPES buffer with BSA (1 mg/ml) containing 100 pM [33P]-H2RLX for 90 min to determine total binding with nonspecific binding defined by incubation with unlabeled H2RLX (1 µM). Labeled sections were washed (2 x 10 min) in HEPES buffer, briefly rinsed in distilled water, and dried with a stream of air. Slides were stored overnight in a vacuum-sealed container with silica gel before commencing the emulsion protocol.

LM-1 hypercoat emulsion (Amersham Pharmacia Biotech, Little Chalfont, UK) was incubated at 42 C (under darkroom conditions) for 1 h and then transferred to a dipping chamber. Each slide was dipped into the emulsion twice, drained, and allowed to dry in a light-proof box overnight. Slides were then transferred into a slide box, wrapped in foil to prevent exposure to light, and stored at 4 C for 3 wk. Before development, slides were equilibrated at room temperature for 1 h. Emulsion was developed in D-19 developer (5 min, Kodak, Rochester, NY), Kodak stop solution (1 min), Kodak fix solution (10 min), and gently rinsed in running water (15 min). Slides were air dried and examined under a dark-field microscope. For histological analysis, slides were stained with Gill’s hematoxylin (distilled water 730 ml, ethylene glycol 250 ml, hematoxylin 2 g, sodium iodate 17.6 g, and glacial acetic acid 20 ml) and eosin (1% solution).

Analysis of RLX, LGR7, and related gene expression

RLX (H1 & H2), LGR7, and VEGF165 gene expression were first assessed by RT-PCR. Analysis of H3RLX was not conducted in this study because there is no known human tissue that expresses H3RLX and hence no positive control cDNA. Total endometrial RNA (1 µg) was used for synthesis of first-strand cDNA in a 20-µl reverse transcription reaction using a reverse transcription kit (Promega Corp., Annandale, NSW, Australia) and random hexamer primers. Between 1 and 2 µg of this cDNA template was used in touch-down PCR (50 µl volume) with 0.2 U Taq DNA polymerase (Promega) and 100 ng/µl human gene-specific oligonucleotide primers (Geneworks Pty. Ltd., Adelaide, Australia). Human LGR7 primers were designed from the human genome sequence (NCBI, accession no. AF190500), whereas all other RLX oligonucleotide primers were obtained from previously published sequences: H1RLX (6), H2RLX (7), and human VEGF165 (20). The RLX and LGR7 oligonucleotide primers used were designed to span intron-exon junctions to avoid genomic DNA contamination, whereas the VEGF165 primers were kindly provided by Dr. Elaine Unemori (Connetics Corp., Palo Alto, CA). All PCRs were carried out in a gene amplifier (PerkinElmer, Norwalk, CT) with an initial denaturation step at 94 C (3 min) and annealing temperatures as shown in Table 1Go. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used in separate PCRs to control for quality and equivalent loading of the cDNA. Aliquots of the PCR products were electrophoresed on 2–4% (wt/vol) agarose gels stained with ethidium bromide and photographed.


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  		TABLE 1. Primers, probes, and conditions used for RT/PCR

 
Real-time PCR was used to quantify H2RLX, LGR7, VEGF165, and VEGF121 gene expression using human gene-specific primers and 6carboxy-fluorescein-labeled probes (Keystone Division, Biosource International, Foster City, CA) designed with the Primer Express computer software (Applied Biosystems, Foster City, CA). The human VEGF primers and probes were kindly provided by Dr. Elaine Unemori (Connetics Corp.). All primers were designed to span introns to avoid contamination with genomic DNA. For each sample, 1 µg total endometrial RNA was reverse transcribed in a 30-µl reaction containing 1 x TaqMan buffer [5.5 mM MgCl2, 500 µM deoxynucleotide triphosphates, 2.5 µM oligo d(T), 0.4 µl RNase inhibitor, and 1.25 U/µl MultiScribe reverse transcriptase (Applied Biosystems)]. For the LGR7 cDNA, 2.5 µM random hexamers replaced the oligo d(T). A second reaction mix using 30 ng total RNA from each sample and a series of endometrium RNA dilutions (100–0.001 ng) was prepared for the endogenous reference 18S rRNA (18S) PCR and to generate the 18S standard curves, respectively. First-strand cDNA synthesis for all samples was carried out simultaneously at 25 C for 10 min, 42 C for 45 min, and 95C for 10 min with a final cooling temperature a 4 C before storage at –20 C. The PCRs were conducted in triplicate using 96-well optical reaction plates (Applied Biosystems) in 25-µl volumes consisting of 1 x TaqMan universal PCR master mix, 0.8 µM forward and reverse primers, 0.4 µM probe, and 2.5 µl cDNA template. An ABI PRISM 7700 sequence detector (Applied Biosystems) was used with the following conditions: 50 C for 10 min and 95 C for 10 min followed by 40 cycles of 95 C for 15 sec and 58 C for 1 min. Negative template samples were included in each plate as controls. The relative CT standard curve method was used in this study, where CT is the cycle number at which DNA amplification is first detected. In the relative standard curve method, specific gene and 18S (endogenous reference) expression are assessed in separate PCRs and the CT values for both genes are related to those of the endogenous reference standard curve. The amount of specific gene expressed in each sample is calculated by dividing the log (gene of interest concentration) by the log (18S concentration) using the following formula: log (gene concentration) = (CT – b/a) where b = y-intercept of the standard curve and a = slope of the standard curve. In this experiment the regression line for the 18S standard curve was y = (–3.398x + 14.235), R2 = 0.998. The same 18S standards were used on every plate, with interplate variation 2.2% (1 ng standard, n = 4 plates).

Materials

H2RLX was kindly provided by Connetics Corp. PRLX (native PRLX) was prepared from pregnant sow ovaries by a method modified from Bullesbach and Schwabe (21) at the Howard Florey Institute by Dr. John Wade, using reverse-phase HPLC and characterized by MALDITOF mass spectrometry (>99% purity). The cAMP-dependent protein kinase was purchased from Promega; [{gamma}-33P]-ATP was obtained from Amersham; and HEPES (BDH), PMSF, BSA, eosin, hematoxylin, and poly-L-lysine were obtained from Sigma (St. Louis, MO). All other chemicals and reagents were of either analytical or laboratory grade.

Statistical analysis

Quantitative autoradiography results were analyzed using one-way ANOVA with a Newman-Keuls posttest. The –log[dissociation constant] (pKD), maximal binding capacity (Bmax), and –log[inhibition constant] (pKi) data were analyzed using a Student’s t test. Data for mRNA concentrations did not show homogeneity of variance and were log transformed before analysis by one-way ANOVA (SPSS 10.0, SPSS Inc., Chicago, IL). The least squares difference method tested for significant differences at the 95% confidence level between stages of the menstrual cycle.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification and characterization of [33P]-H2RLX binding by quantitative autoradiography

Changes in binding of [33P]-H2RLX with the phases of the menstrual cycle. Slide-mounted sections of uterus from the seven phases of the menstrual cycle were incubated with [33P]-H2RLX to determine receptor binding levels and any changes that may occur during the menstrual cycle. Initial studies were conducted blind with a variety of sections from different phases of the cycle and showed strong binding in some sections but not others. Sections with high levels of binding were subsequently shown to be from the secretory phase of the cycle and those with little binding from the proliferative phase (Fig. 1AGo). Systematic studies of uterine sections from the EP, MP, and LP phases showed very little [33P]-H2RLX binding (Fig. 1BGo), whereas those from the ES, MS, and LS showed a large amount of binding, mainly confined to the endometrium (Fig. 1BGo). Binding levels were markedly increased in the ES and MS phases and then declined in the LS and M phase, although the levels were still higher than in the EP stage.



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  		FIG. 1. Autoradiographic localization of [33P]-H2RLX binding to slide-mounted sections of human uterus at seven different phases of the menstrual cycle. A, Left panels are representative slides of the total, and right panels of nonspecific [33P]-H2RLX binding from each of the phases of the menstrual cycle. B, Levels of specific binding determined by quantitative autoradiography from each of the phases in the menstrual cycle (mean ± SEM; n) are shown. Binding in the ES phase was significantly different (**, P < 0.001) from all proliferative phases (EP, MP and LP), and binding in LS was significantly different (*, P < 0.05) from EP. Binding decreased significantly from the ES to LS phase (*, P < 0.05).

 
Characterization of [33P]-H2RLX binding in the proliferative and secretory phases. The saturation and competition binding properties of [33P]-H2RLX to sections from the early-midproliferative (E-MP) and early-midsecretory (E-MS) were studied to determine whether the changes in intensity are related to an alteration in receptor number or affinity. Binding to sections from both the E-MP and E-MS phases was saturable to a single population of binding sites (Fig. 2AGo), which displayed no significant difference in the binding affinity (E-MP pKD 9.16 ± 0.17, n = 4; E-MS pKD 9.16 ± 0.06, n = 4). However, the number of binding sites in E-MS was more than 10 times higher (Bmax 160 ± 32; n = 4) than in the E-MP sections (Bmax 15 ± 4; n = 4; P < 0.005).



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  		FIG. 2. RLX receptor characterization determined using quantitative auto-radiography of [33P]-H2RLX binding to slide-mounted sections of human uterus. Sections of uterus tissue were obtained from the proliferative (E-MP; left panels) and secretory stages (E-MS; right panels) of the cycle. A, Saturation binding isotherms show that the binding of [33P]-H2RLX is saturable and to a single population of sites. B, Competition for [33P]-H2RLX binding by H2RLX {blacksquare} and PRLX {blacktriangledown}.

 
The ability of H2RLX and PRLX to compete for [33P]-H2RLX binding was examined in competition studies in uterine sections to determine whether other RLX analogs competed for binding and whether the characteristics of binding altered with the phase of the cycle. pIC50 values were calculated from competition curves fitted using Prism and pKi values calculated using the equation of Cheng and Prusoff (22). Sections from the E-MP showed that H2RLX (pKi 8.0 ± 0.06, n = 4) had a significantly higher affinity for the RLX receptor in the endometrium than PRLX (pKi 7.4 ± 0.21, n = 4, P < 0.05). The same pattern was observed in the E-MS, with H2RLX (pKi 8.3 ± 0.11, n = 4) having a significantly higher affinity for the receptor than PRLX (pKi 7.3 ± 0.08, n = 4, P < 0.0005; Fig. 2BGo). Although the density of RLX binding was much higher in the E-MS phase, there was no significant difference between the affinity of either H2RLX or PRLX determined in the E-MP or E-MS phase.

Localization of the RLX receptor in uterine sections using emulsion autoradiography

Low levels of silver grains appeared over the endometrium of sections from the E-MP phase with no significant silver grains overlying the myometrium. In contrast, in sections from E-MS, an abundance of silver grains was highly localized to sites in the endometrium, with nonspecific levels of silver grains associated with the myometrium. Comparison of corresponding dark-field and light-field images showed that the silver grains in the endometrium were localized to the glandular epithelial cells (Fig. 3AGo) and the luminal epithelium (Fig. 3BGo).



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  		FIG. 3. Qualitative autoradiographic localization of [33P]-H2RLX binding using photographic emulsion on slide-mounted sections of human uterus obtained during the E-MS phase. Light-field (LF) and dark-field (DF) images of myometrium (M) and endometrium (E) (A) demonstrate silver grains abundant in the glandular epithelial cells of the endometrium (x2 magnification) and in the endometrial luminal epithelium (B) (x10 magnification).

 
Analysis of RLX, LGR7, and related gene expression

H1RLX gene transcripts were only weakly expressed in two samples taken at the proliferative phase of the cycle by RT-PCR (Fig. 4Go). Gene transcripts of H2RLX were detected at relatively weak but consistent levels in both the proliferative and secretory stages of the cycle and appear to be the predominant form of RLX in the human endometrium. Of the two bands observed, the 445-bp band corresponds to the expected product for H2RLX, whereas the 330-bp band represents an unrelated fragment of DNA. H2RLX was highly expressed in the corpus luteum as expected (Fig. 4Go); however, no direct comparison can be made between the endometrium and the ovarian samples because different amounts of RNA were used in the first-strand cDNA synthesis.



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  		FIG. 4. Expression of RLX (H1 and H2), LGR7, and VEGF165 mRNA using RT-PCR on RNA extracted from the human endometrium at various phases of the menstrual cycle. Shown is a molecular weight marker (left lane), followed by samples from separate patients in the proliferative phase (lanes 1–5), ES phase (lanes 6–8), MS phase (lanes 9–11), LS phase (lanes 12–14), and M phase (lanes 15–16) of the cycle. RNA from human prostate was used as a positive control for H1RLX PCR, whereas RNA from human corpus luteum and THP-1 cells were used as positive controls for H2RLX PCR and LGR7 PCR, respectively (all lane 17). Because the endometrium is a major source of VEGF expression, an additional positive control was not included for the VEGF PCR. GAPDH products were used as controls for quality and equal loading of the cDNA (lanes 1–16). Water replaced the cDNA in negative control reactions for each PCR (lane 18).

 
An LGR7 gene transcript (634 bp), representing the full-length, functional version of LGR7, was detected in the human endometrium, with stronger expression observed in those samples obtained from the secretory phase of the cycle (Fig. 4Go). The RT-PCR primers for LGR7 were designed to measure only the full-length and not the truncated, alternatively spliced version of the RLX receptor. VEGF165 gene expression was also higher in the secretory phase of the cycle, with seemingly stronger expression observed in the LS endometrium (Fig. 4Go). An additional positive control was not included because the endometrium is generally considered to be a major source of VEGF.

Quantitative analysis by real-time PCR confirmed that there was a significant (P < 0.01 ANOVA) increase in the endometrial LGR7 mRNA concentrations in the early secretory phase, compared with all other stages of the cycle (Fig. 5AGo). Although LGR7 gene expression decreased in the MS endometrium, mRNA concentrations remained significantly (P < 0.05) higher in the MS, LS, and M phases, compared with the proliferative phase of the cycle. This pattern of LGR7 gene expression paralleled that of RLX binding in the endometrium assessed by quantitative autoradiography (Fig. 1BGo). The real-time PCR for H2RLX demonstrated a low level of gene expression in the endometrium with CT values of between 38 and 40 (data not shown). Thus, it was not possible to show differences for H2RLX between the phases of the cycle.



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  		FIG. 5. LGR7 (A), VEGF165 (B), and VEGF121 (C) mRNA concentrations in the endometrium at five phases of the menstrual cycle. Data are the mean ± SEM for RNA extracted from n = 4–5 tissues, gene/18S ratio x 103. a, Significantly (P < 0.05) higher than all other phases; b, significantly (P < 0.05) higher than the proliferative phase; c, significantly (P < 0.05) higher than the proliferative phase and early secretory phase.

 
A comparison of VEGF165 and VEGF121 mRNA concentrations in the endometrium throughout the cycle showed an identical pattern of gene expression for both isoforms (Fig. 5Go, B and C). Concentrations were low in the proliferative phase of the cycle and did not differ significantly (P < 0.05), compared with ES and MS endometrium. Expression of both VEGF isoforms increased significantly (P < 0.05) in the LS phase and remained high in the M phase.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The aim of the present study was to investigate the sites of expression and the characteristics of the RLX receptor in the human uterus at different phases throughout the menstrual cycle. Whereas binding sites for biotinylated PRLX have previously been identified in the human uterus (4), until recently no studies have been carried out to identify changes in RLX receptor expression during the menstrual cycle. The RLX peptide has previously been identified in human endometrial tissue in the secretory phase; however, no RLX peptide was detectable in the proliferative phase (23). RLX has been shown to differentially influence VEGF secretion in isolated uterine cells taken from both the proliferative and secretory phases (11), although it was unclear whether the differential regulation occurs at the level of the receptor or the signaling cascade.

The results reported in this study indicate that expression of the RLX receptor varies in a cyclical manner through the menstrual cycle. A low level of receptor binding was demonstrated during the proliferative stages with a dramatic increase in binding at the time of ovulation. The high levels of binding were maintained during the ES (d 15–18 of a standardized 28-d cycle) and MS phase (d 19–24) and then decreased through the LS and M phases (d 25–28). Results from the gene expression studies supported the autoradiography and showed a 13-fold increase in LGR7 mRNA after ovulation, which subsequently decreased in the MS to LS and M stages of the cycle. The more rapid decline in the LGR7 mRNA, compared with the binding, probably reflects a more rapid turnover of RNA than receptor protein. This is the first study to identify a cyclic expression of the RLX receptor in the human uterus. Two studies have appeared while our work was under review. Immunohistochemical staining of endometrial sections from three patients showed little if any staining in sections from patients in the proliferative or ES phase but intense staining in one patient in the MS phase (24). Although the other study (25) reported no consistent regulation of LGR-7 mRNA during the phases of the menstrual cycle, the expression levels are expressed as a ratio with GAPDH, which is known to be sex steroid dependent (26). In the present study, the quantitative PCR was carried out using 18S RNA as the standard. Given our finding that RLX receptor is elevated in the secretory phase and the observation that plasma RLX levels increase from d 5 to peak at d 10 post ovulation (9), it is possible that a role exists for RLX in the process of embryo implantation. Analysis of gene expression of the RLX peptides indicated that the endometrium is not a major source of circulating RLX, with low to undetectable levels of RLX mRNA present throughout all stages of the menstrual cycle. Our data are in agreement with recent work (11) that demonstrated expression of RLX in the human endometrium using a nested PCR method. The major source of circulating RLX is the corpus luteum because it is highly expressed in this tissue (10), high levels of the hormone are found in the ovarian vein (27), and women without ovaries who become pregnant by oocyte donation have no circulating RLX (28, 29). Local production of RLX from the endometrium may therefore play a significant role in the establishment or maintenance of early pregnancy.

The binding characteristics of the RLX receptor remained unaltered between the proliferative and secretory phases, with the pKD values for the [33P]-H2RLX ligand and the pKi values for H2RLX and PRLX being not significantly different with the changing stages of the cycle. The values determined were comparable with those obtained with previous binding studies using THP-1 cells (30), which endogenously express the human RLX receptor (31), or human uterine cells (32, 33), indicating that THP-1 cells and the uterus express the same receptor. The consistency in the binding characteristics between the different menstrual cycle phases indicates that the variations in RLX function during the menstrual cycle, such as its potential influence on VEGF secretion (11), are not due to changes in receptor conformation or affinity of the RLX receptors expressed. RLX receptor expression in the endometrium during the secretory phase was confined specifically to glandular and luminal epithelial cells. The binding of [33P]-H2RLX was appropriate for binding to the RLX receptor because it was competitively inhibited by both H2RLX and PRLX with high affinity. In contrast to previous studies that showed binding of biotinylated PRLX to blood vessels and myometrium in addition to glandular and luminal epithelium (4), the present studies provided no evidence for binding of [33P]-H2RLX to these tissues. The differing results may relate to the concentrations of labeled RLX used in the two studies. In the present study we used [33P]-H2RLX at a concentration of 100 pM, similar to the known pKD value for RLX at the receptor (19), whereas the study using biotinylated PRLX used a concentration of approximately 0.7 µM, several orders of magnitude higher than the pKD. It is possible that the use of high labeling concentrations could result in binding to low affinity sites such as the related LGR8 receptor, which is known to bind RLX (1). However, the lack of binding to stromal cells was surprising, given the demonstration of functional responses in these cells in culture (5, 11, 14). The reason for the difference may relate to the different conditions experienced by cells in situ and those in culture because human endometrial stromal cells fail to produce a cAMP response to RLX when first isolated, but the response appears gradually over the first 11 d of culture and then increases dramatically from 11–30 d (34). As with stromal cells, studies with human myometrial cell lines (PHM1–41) isolated from term pregnant myometrium show clear evidence of RLX stimulated cAMP accumulation indicating the presence of RLX receptors (35). This may also relate to the expression of relaxin receptors in an immortalized cell line under cell culture conditions. In contrast, functional studies of the effects of relaxin in the human uterus show that human myometrium is much less able to respond in vitro to relaxins than either pig or rat myometrium. Porcine relaxin had little relaxant effect on myometrium from pregnant or nonpregnant women (36). Later studies with recombinant H2RLX showed little or no relaxant effect on human myometrium excised at hysterectomy or at cesarean section (37) or on uterine fundus or isthmus at term (38). Studies of spontaneous contractions of human myometrium demonstrated small reductions in amplitude to relaxin-rich extracts of corpus luteum (39) or of amplitude and frequency to H2RLX in some estrogen-primed tissues (37). This is in direct contrast to studies in pig myometrium or rat uterus where complete inhibition of contraction was observed with H2RLX, PRLX and H1RLX (37, 38, 40, 41). The lack of significant [33P] H2RLX binding to relaxin receptors in the human myometrium reported here is in accord with the poor relaxant responses to relaxin observed in this tissue.

Immunohistochemical studies demonstrated RLX in endometrium in the secretory but not in the proliferative phase nor in myometrium from either phase (23). The hormone has also been localized to decidualized stromal cells in the LS phase and early pregnancy (11). Recent studies have established that RLX is synthesized in the endometrium because RLX mRNA was present in cultured endometrial stromal and glandular epithelial cells with stronger bands seen in cells from the secretory phase and immunoreactive RLX was detected in culture medium bathing the cells (11). Because there was a lack of evidence for RLX receptors associated with the endometrial vasculature, our study does not support a direct role for RLX in regulating endometrial vascular function. However, RLX does increase VEGF expression in glandular epithelial and stromal cells from the secretory phase (11), suggesting that it may have a role in endometrial vascularization. The significant increase in RLX binding in the ES phase, as well as the retention of the elevated binding throughout the secretory phase, suggests a role for RLX in implantation. Support for this hypothesis comes from a study showing that high levels of RLX produced in granulosa lutein cell cultures are associated with 100% implantation success rates with in vitro fertilization, whereas low levels resulted in an implantation rate of 13% (42). Although this would indicate that the production of RLX is linked to successful implantation, circulating RLX is clearly not obligatory for this process because implantation can occur in women without ovaries (28, 29). This may indicate that local RLX may be important, that it is a marker for other factors important in implantation, or merely that it is a marker of oocyte quality. VEGF is known to have an important role in the proliferation and vascularization of the uterus lining, and although relaxin has been found to stimulate VEGF secretion in glandular epithelial and stromal cells (11), the VEGF responsible for uterus growth has been identified in intravascular neutrophils rather than from the glandular epithelial cells (43). The increase in VEGF165 and VEGF121 gene expression during the MS to LS phases supports previous reports showing an increase in VEGF mRNA during this stage of the cycle (44).

In conclusion, we have localized binding sites for RLX in the human uterus and have demonstrated that they are primarily confined to the glandular and luminal epithelium in the secretory stage of the cycle. Furthermore, the levels of RLX receptor expression change dramatically during the different phases of the menstrual cycle. The binding characteristics of the receptor were appropriate for binding to the RLX receptor (LGR7) and were unchanged with differing expression levels. Thus, any difference in RLX function between the phases is a reflection of changes in the intracellular properties of the receptor and not due to changes in receptor characteristics. The location and regulation of RLX receptor expression that occur during the menstrual cycle suggest a role in the implantation process, although further studies are needed to confirm this hypothesis.


   Acknowledgments
 
The authors thank Drs. Caroline Gargett and Gareth Weston for technical help and advice and provision of some human uterus samples used in the study. Thanks are also due to Sisters Nancy Taylor and Nicki Sam for the collection of tissue samples and to the various gynecological surgeons affiliated with Monash Medical Centre who provided subjects for the study. We thank Chongxin Zhao for her assistance with the RT-PCR experiments. The authors are particularly grateful to Dr. Elaine Unemori for providing FAM-labeled probes and advice on the real-time PCR component of the study. We also thank Gillian Bryant-Greenwood and Lily Tashima for the human H1 and H2 real-time PCR primers and probe sequences. Dr. John Wade (Howard Florey Institute) kindly provided purified native PRLX, and Connetics Corp. provided recombinant human (B29) RLX.


   Footnotes
 
This work was supported in part by a National Health and Medical Research Council Block Grant to the Howard Florey Institute (Reg Key 983001) and an Australian Research Council (ARC) Linkage Grant to Laura Parry, Roger Summers, and Geoff Tregear (LP0211545). P.A.W.R. is a Principal Research Fellow of the National Health and Medical Research Council of Australia (Fellowship Grant 143805). L.J.P. is an ARC QEII Fellow, and C.S.S. is a recipient of an ARC Postdoctoral (Industry) Fellowship.

Abbreviations: Bmax, Maximal binding capacity; CT, cycle number at which DNA amplification is first detected; E-MP, early-midproliferative; E-MS, early-midsecretory; EP, early proliferative; ES, early secretory; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H2RLX, recombinant human gene 2 RLX; LP, late proliferative; LS, late secretory; M, menstrual; MP, midproliferative; MS, midsecretory; pKD, –log[dissociation constant]; pKi, –log[inhibition constant]; PMSF, phenylmethylsulfonylfluoride; PRLX, porcine RLX; RLX, relaxin; 18S, 18S rRNA; VEGF, vascular endothelial growth factor.

Received May 8, 2003.

Accepted April 5, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hsu SY, Nakabayashi K, Nishi S, Kumagai J, Kudo M, Sherwood OD, Hsueh AJ 2002 Activation of orphan receptor by the hormone relaxin. Science 25:637–638
  2. Hsu SY, Kudo M, Chen T, Nakabayashi K, Bhalla A, van der Spek PJ, van Duin M, Hsueh AJ 2000 The three subfamilies of leucine-rich repeat-containing G protein-coupled receptors (LGR): identification of LGR6 and LGR7 and the signaling mechanism for LGR7. Mol Endocrinol 14:1257–1271[Abstract/Free Full Text]
  3. Fei DT, Gross MC, Lofgren JL, Mora-Worms M, Chen AB 1990 Cyclic AMP response to recombinant human relaxin by cultured human endometrial cells—a specific and high throughput in vitro bioassay. Biochem Biophys Res Commun 170:214–222[CrossRef][Medline]
  4. Kohsaka T, Min G, Lukas G, Trupin S, Campbell ET, Sherwood OD 1998 Identification of specific relaxin-binding cells in the human female. Biol Reprod 59:991–999[Abstract/Free Full Text]
  5. Einspanier A, Muller D, Lubberstedt J, Bartsch O, Jurdzinski A, Fuhrmann K, Ivell R 2001 Characterization of relaxin binding in the uterus of the marmoset monkey. Mol Hum Reprod 7:963–970[Abstract/Free Full Text]
  6. Hudson P, Haley M, Cronk M, Crawford R, Haralambidis J, Tregear G, Shine J, Niall H 1983 Structure of a genomic clone encoding biologically active human relaxin. Nature 301:628–631[CrossRef][Medline]
  7. Hudson P, John M, Crawford R, Haralambidis J, Scanlon D, Gorman J, Tregear G, Shine J, Niall H 1984 Relaxin gene expression in human ovaries and the predicted structure of a human preprorelaxin by analysis of cDNA clones. EMBO J 3:2333–2339[Medline]
  8. Bathgate RA, Samuel CS, Burazin TC, Layfield S, Claasz AA, Reytomas IG, Dawson NF, Zhao C, Bond CP, Summers RJ, Parry LJ, Wade JD, Tregear GW 2002 Human relaxin gene 3 (H3) and the equivalent mouse relaxin (M3) gene. Novel members of the relaxin peptide family. J Biol Chem 277:1148–1157[Abstract/Free Full Text]
  9. Stewart DR, Celniker AC, Taylor Jr CA, Cragun JR, Overstreet JW, Lasley BL 1990 Relaxin in the peri-implantation period. J Clin Endocrinol Metab 70:1771–1773[Abstract/Free Full Text]
  10. Ivell R, Hunt N, Khan-Dawood F, Dawood MY 1989 Expression of the human relaxin gene in the corpus luteum of the menstrual cycle and in the prostate. Mol Cell Endocrinol 66:251–255[CrossRef][Medline]
  11. Palejwala S, Tseng L, Wojtczuk A, Weiss G, Goldsmith LT 2002 Relaxin gene and protein expression and its regulation of procollagenase and vascular endothelial growth factor in human endometrial cells. Biol Reprod 66:1743–1748[Abstract/Free Full Text]
  12. Bryant-Greenwood GD, Rutanen EM, Partanen S, Coelho TK, Yamamoto SY 1993 Sequential appearance of relaxin, prolactin and IGFBP-1 during growth and differentiation of the human endometrium. Mol Cell Endocrinol 95:23–29[CrossRef][Medline]
  13. Telgmann R, Gellersen B 1998 Marker genes of decidualization: activation of the decidual prolactin gene. Hum Reprod Update 4:472–479[Abstract/Free Full Text]
  14. Bell SC, Jackson JA, Ashmore J, Zhu HH, Tseng L 1991 Regulation of insulin-like growth factor-binding protein-1 synthesis and secretion by progestin and relaxin in long term cultures of human endometrial stromal cells. J Clin Endocrinol Metab 72:1014–1024[Abstract/Free Full Text]
  15. Tseng L, Zhu HH, Mazella J, Koistinen H, Seppala M 1999 Relaxin stimulates glycodelin mRNA and protein concentrations in human endometrial glandular epithelial cells. Mol Hum Reprod 5:372–375[Abstract/Free Full Text]
  16. Unemori EN, Erikson ME, Rocco SE, Sutherland KM, Parsell DA, Mak J, Grove BH 1999 Relaxin stimulates expression of vascular endothelial growth factor in normal human endometrial cells in vitro and is associated with menometrorrhagia in women. Hum Reprod 14:800–806[Abstract/Free Full Text]
  17. Noyes R, Hertig A, Rock J 1950 Dating the endometrial biopsy. Fertil Steril 1:3–25
  18. Weston GC, Haviv I, Rogers PA 2002 Microarray analysis of VEGF-responsive genes in myometrial endothelial cells. Mol Hum Reprod 8:855–863[Abstract/Free Full Text]
  19. Tan YY, Wade JD, Tregear GW, Summers RJ 1999 Quantitative autoradiographic studies of relaxin binding in rat atria, uterus and cerebral cortex: characterization and effects of oestrogen treatment. Br J Pharmacol 127:91–98[CrossRef][Medline]
  20. Houck KA, Ferrara N, Winer J, Cachianes G, Li B, Leung DW 1991 The vascular endothelial growth factor family: identification of a fourth molecular species and characterization of alternative splicing of RNA. Mol Endocrinol 5:1806–1814[Abstract/Free Full Text]
  21. Büllesbach EE, Schwabe C 1985 Naturally occurring porcine relaxin and large-scale preparation of the B29 hormone. Biochemistry 24:7717–7722[CrossRef][Medline]
  22. Cheng YC, Prusoff WH 1973 Relationship between the inhibition constant Ki and the concentration of inhibitor which causes 50% inhibition (IC50) of an enzyme reaction. Biochem Pharmacol 22:3099–3108[CrossRef][Medline]
  23. Yki-Jarvinen H, Wahlstrom T, Seppala M 1983 Immunohistochemical demonstration of relaxin in the genital tract of pregnant and non-pregnant women. J Clin Endocrinol Metab 57:451–454[Abstract/Free Full Text]
  24. Ivell R, Balvers M, Pohnke Y, Telgmann R, Bartsch O, Milde-Langosch K, Bamberger A-M, Einspanier A 2003 Immunoexpression of the relaxin receptor LGR7 in breast and uterine tissues of humans and primates. Reprod Biol Endocrinol 1:114[CrossRef][Medline]
  25. Luna JJ, Riesewijk A, Horcajadas JA, de van Os R, Dominguez F, Mosselman S, Pellicer A, Simon C 2004 Gene expression pattern and immunoreactive protein localisation of LGR7 receptor in human endometrium throughout the menstrual cycle. Mol Hum Reprod 10:85–90[Abstract/Free Full Text]
  26. Wu X, Englund K, Lindblom B, Blanck A 2003 mRNA expression of often used house-keeping genes and the relation between RNA and DNA are sex-steroid-dependent parameters in human myometrium. Gynecol Obstet Invest 55:225–230[CrossRef][Medline]
  27. Weiss G, O’Byrne EM, Steinetz BG 1976 Relaxin: a product of the human corpus luteum of pregnancy. Science 194:948–949[Abstract/Free Full Text]
  28. Emmi AM, Skurnick J, Goldsmith LT, Gagliardi CL, Schmidt CL, Kleinberg D, Weiss G 1991 Ovarian control of pituitary hormone secretion in early human pregnancy. J Clin Endocrinol Metab 72:1359–1363[Abstract/Free Full Text]
  29. Johnson MR, Abdalla H, Allman AC, Wren ME, Kirkland A, Lightman SL 1991 Relaxin levels in ovum donation pregnancies. Fertil Steril 56:59–61[Medline]
  30. Tan YY, Bond CP, Wade JD, Tregear GW, Summers RJ 2000 Bioassays for relaxin and relaxin-related peptides. In: Tregear GW, ed. Relaxin 2000. Broome, WA, Australia: Kluwer Academic Publishers; 271–272
  31. Parsell DA, Mak JY, Amento EP, Unemori EN 1996 Relaxin binds to and elicits a response from cells of the human monocytic cell line, THP-1. J Biol Chem 271:27936–27941[Abstract/Free Full Text]
  32. Palejwala S, Stein D, Wojtczuk A, Weiss G, Goldsmith LT 1998 Demonstration of a relaxin receptor and relaxin-stimulated tyrosine phosphorylation in human lower uterine segment fibroblasts. Endocrinology 139:1208–1212[Abstract/Free Full Text]
  33. Osheroff PL, King KL 1995 Binding and cross-linking of 32P-labeled human relaxin to human uterine cells and primary rat atrial cardiomyocytes. Endocrinology 136:4377–4381[Abstract]
  34. Tseng L, Lane B 1995 Role of relaxin in the decidualization of human endometrial cells. In: MacLennan AH, Tregear G, Bryant-Greenwood G, eds. Relaxin ’94. Adelaide, SA, Australia: World Scientific Publishing Co. Pte. Ltd.; 325–338
  35. Dodge KL, Sanborn BM 1998 Evidence for inhibition by protein kinase A of receptor/G {alpha}(q)/phospholipase C (PLC) coupling by a mechanism not involving PLCß2. Endocrinology 139:2265–2271[Abstract/Free Full Text]
  36. MacLennan AH, Grant P, Ness D, Down A 1986 Effect of porcine relaxin and progesterone on rat, pig and human myometrial activity in vitro. J Reprod Med 31:43–49[Medline]
  37. MacLennan AH, Grant P 1991 Human relaxin. In vitro response of human and pig myometrium. J Reprod Med 36:630–634[Medline]
  38. Petersen LK, Svane D, Uldbjerg N, Forman A 1991 Effects of human relaxin on isolated rat and human myometrium and uteroplacental arteries. Obstet Gynecol 78:757–762[Medline]
  39. Szlachter N, O’Byrne E, Goldsmith L, Steinetz BG, Weiss G 1980 Myometrial inhibiting activity of relaxin-containing extracts of human corpora lutea of pregnancy. Am J Obstet Gynecol 136:584–586[Medline]
  40. MacLennan AH, Green RC, Grant P, Nicolson R 1986 Ripening of the human cervix and induction of labor with intracervical purified porcine relaxin. Obstet Gynecol 68:598–601[Medline]
  41. MacLennan AH, Grant P, Bryant-Greenwood G 1995 hRLX-1. In vitro response of human and pig myometrium. J Reprod Med 40:703–706[Medline]
  42. Stewart DR, VandeVoort CA 1999 Relaxin secretion by human granulosa cell culture is predictive of in vitro fertilization-embryo transfer success. Hum Reprod 14:338–344[Abstract/Free Full Text]
  43. Gargett CE, Lederman F, Heryanto B, Gambino LS, Rogers PAW 2001 Focal vascular endothelial growth factor correlates with angiogenesis in human endometrium. Role of intravascular neutrophils. Hum Reprod 16:1065–1075[Abstract/Free Full Text]
  44. Sugino N, Kashida S, Karube-Harada A, Takiguchi S, Kato H 2002 Expression of vascular endothelial growth factor (VEGF) and its receptors in human endometrium throughout the menstrual cycle and in early pregnancy. Reproduction 123:379–387[Abstract]



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