Molecular Human Reproduction, Vol. 10, No. 2, pp. 85-90, 2004
© European Society of Human Reproduction and Embryology 2004
Gene expression pattern and immunoreactive protein localization of LGR7 receptor in human endometrium throughout the menstrual cycle
1Foundation of the Instituto Valenciano de Infertilidad, and 2Department of Pediatrics, Obstetrics and Gynecology, Valencia University School of Medicine, Valencia, Spain, 3Instituto de Ciencias en Reproducción Humana, León, Guanajuato, México and 4NV Organon, Departments of Target Discovery and Pharmacology, Oss, The Netherlands
5 To whom correspondence should be addressed at: Plaza de la Policía Local 3, 46015 Valencia, Spain. e-mail: csimon{at}interbook.net
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Relaxin (RLX) is a pregnancy-associated polypeptide hormone. In non-pregnant women, the peak of circulating relaxin coincides with the window of endometrial receptivity and both in vivo and in vitro experiments showed that it plays a role in the decidualization process. Recently, two receptors, LGR7 and LGR8, have been identified as high affinity receptors for relaxin. Here we describe LGR7 mRNA and protein expression in human endometrium using semi-quantitative and quantitative fluorescent PCR (Q-PCR) and immunohistochemical analyses. Three different experimental designs were used. First, endometrial biopsies from five different phases of the menstrual cycle were analysed. Secondly, we assessed the early luteal phase in more detail. Finally we analysed the expression at LH+2 (2 days after the natural LH surge, pre-receptive endometrium) versus LH+7 (receptive endometrium) within the same menstrual cycle from the same patient to avoid inter-cycle or inter-person variations in gene expression. Our results indicate that there is no consistent regulation of LGR7 mRNA expression, neither during the menstrual cycle nor during the early–mid-luteal phase. In general, we observed a large degree of variation in LGR7 mRNA expression levels between patients. LGR7 immunoreactive protein was identified in all stages of the menstrual cycle. LGR7 protein was localized in both the epithelial and the stromal compartments, except for the mid-luteal phase when the expression was restricted to the endometrial epithelium. We conclude that no consistent regulation of LGR7 mRNA expression can be detected in human endometrium during the menstrual cycle.
Key words: Key words: endometrium/LGR7 receptor/relaxin
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Introduction |
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Relaxin is a peptide hormone produced by the corpus luteum during the luteal phase of the menstrual cycle and during the first trimester of pregnancy. It is composed of two peptide chains, A and B, of 24 and 29 amino acids respectively, linked by disulphide bridges. Relaxin belongs structurally to a family of closely related protein hormones which includes, besides insulin and relaxin, insulin like-growth factor-I (IGF-I), IGF-II, the relaxin factor (RLF/INSL3) and several novel relaxin/insulin-like factors 2 (INSL4) and 1 (INSL6) (Ivell and Bathgate, 2002).
Relaxin has been associated with a wide range of functions related to pregnancy such as parturition and lactation (Telgmann et al., 1998). In animal models such as pigs and rats, relaxin softens the cervix (ripening), inhibits uterine contractions, and relaxes the pubic symphysis. In vitro studies with human cervical stromal cells indicate that porcine relaxin increases proteinase activity, inducing an enzymatic breakdown of collagen, which constitutes in part the cellular basis of the cervical softening before labour (Hwang et al., 1996). A marmoset monkey study using labelled relaxin showed an increased binding pattern reaching a peak during the secretory phase, suggesting the involvement of specific relaxin receptors in implantation and early pregnancy (Einspanier et al., 2001). Functionally, it was shown that human endometrial cells in vitro treated with relaxin respond with an increase in VEGF secretion in a dose-dependent manner (Unemori et al., 1999). In addition to VEGF, IGF-binding protein 1 (Tseng et al., 1992), and prolactin (Huang et al., 1987) are also up-regulated by relaxin.
Though relaxin is a well-studied hormone, very little was known about its receptor. Recently, Hsu et al. (2002) showed that LGR7 and LGR8, two leucine-rich repeat-containing G-protein coupled receptors, in vitro bind relaxin as a high affinity ligand, resulting in cAMP signalling. LGR7 has the highest affinity for relaxin and the potency of LGR8 is somewhat lower. Also it was shown that RLF/INSL3 is a specific and high affinity ligand for LGR8 (Kumagai et al., 2002). The LGR7 gene was first cloned in 2000 and was localized to chromosome 4q32 (Hsu et al., 2000). Two different splice variants of LGR7, differing in the N terminus of the coding region, are described. The long form of LGR7 (1) is 3759 nucleotides long and encodes an open reading frame (ORF) of 759 amino acids with a calculated molecular mass of 87 kDa; whereas the short LGR7 (2) variant is 34 amino acids shorter. LGR7 contains an N-terminal low density lipoprotein (LDL) receptor cysteine-rich motif followed by leucine-rich repeats and a seven transmembrane region followed by a unique C terminal intracellular tail (Braun et al., 1991; Ji et al., 1997).
Previous studies of peripheral relaxin dynamics together with the fact that RT–PCR analysis on total human cDNA reveals that LGR7 and LGR8 are expressed in the uterus (Hsu et al., 2002) suggest a possible role for the relaxin system in human reproductive physiology and endometrial receptivity. Therefore, the aim of the present work was to investigate the gene expression pattern and protein localization of LGR7 receptor in human endometrium throughout the menstrual cycle with particular interest during the implantation window.
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Human tissue collection
Endometrial biopsies were obtained from normal, fertile, cycling women during their menstrual cycle using a Pipelle catheter (Genetics, Belgium). Women were recruited after written informed consent and the project was approved by the institutional review board on the use of human subjects in research at the Instituto Valenciano de Infertilidad. For the samples obtained randomly during the menstrual cycle, a small portion of each specimen was formalin-fixed and paraffin-embedded. Endometrial dating was determined histologically by an experienced pathologist according to the criteria of Noyes et al. (1950). These samples were categorized in five groups: early– mid-proliferative (days 5–8, n = 2), late proliferative (day 9–14, n = 3), early secretory (day 15–18, n = 2), mid-secretory (days 19–23, n = 3) and late secretory (days 24–28, n = 3). For the detailed study of the early luteal phase, endometrial biopsies were collected during the formation of a receptive endometrium 1–7 days after determination of the LH surge: LH+1 (n = 4), LH+2 (n = 13), LH+3 (n = 5), LH+5 (n = 1) and LH+7 (n = 18) and histologically dated. In a third experiment, to minimize inter-individual variations two endometrial biopsies were collected from the same patient (n = 13), in the same menstrual cycle at LH+2 and LH+7. The fold-change of LGR7 expression was calculated based on the identified normalized expression value of the LH+7 sample divided by the normalized expression value of the LH+2 sample in the same patient.
RNA isolation and cDNA generation
Endometrial biopsies were snap-frozen in liquid nitrogen and stored at –70°C until further processing. Total RNA was isolated using TRIzol (Life Technololgies, Inc.), as described previously (Riesewijk et al., 2003). RNA quality was verified either by Agilent 2100 bioanalyzer (Agilent technologies) analysis, or by spectrophotometry (SmartSpec 3000; Biorad, Spain), giving rise to OD 260/280 ratios between 1.5 and 1.9. Human liver RNA was kindly donated by Dr P.Sanz, La Princessa Hospital, Madrid) and THP1 cell line (ECCC88081201) was kindly provided by Dr E.Gomez, Universidad Complutense, Madrid.
One microgram of total RNA was used as template together with oligo (dT)15 primer (Promega, cat. no. c1101) and 500 ng random hexamer primers. Samples were mixed and incubated at 70°C followed by an incubation on ice. Reverse transcriptase (Superscript II cat. no. 18064-014 or MLV (Advantage RT for PCR Kit; BD Bioscience, USA) was added, together with first strand buffer, 0.01 mol/l dithiothreitol and dNTP. The tubes were mixed and incubated for 1 h at 37°C. The reaction was stopped by heat inactivation for 5 min at 94°C.
(Q)-PCR conditions
Primer sequences used in the different experiments are given in Table I. For semi-quatitative PCR the following conditions were used: LGR7 and GAPDH primers 0.25 µmol/l, MgCl2 1.1 mmol/l, and dNTP 0.1 mmol/l in a final volume of 25 µl.
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Q-PCR was performed using the SYBR Green PCR Master Mix and the universal thermal cycling parameters as indicated by the manufacturer. In the menstrual cycle analyses, 250 ng of cDNA was used and the assays were performed on a LightCycler (Roche Diagnostics, GMBH, Germany). For the LH+1 to LH+7 analyses, cDNA equivalent to 10 ng of RNA was amplified using the ABI Prism 7900 sequence detection system (PE Biosystems). Quantification was done by the standard curve method according to the User Bulletin 2, ABI Prism 7700 Sequence Detection System (December 11, 1997) from PE Applied Biosystems. Data are shown as relative values after normalization with the housekeeping gene GAPDH or Capping protein. To obtain the LH+2 versus LH+7 ratio in single patients the LH+7 normalized value was divided by the LH+2 normalized value from the same patient. Only when this resulted in a >2-fold change in LGR7 expression was this scored as significant regulation.
Immunohistochemical analysis
Human endometrial tissues corresponding to days 6, 11, 16, 22 and 28 were obtained from normal cycling patients, formalin-fixed, paraffin-embedded and mounted on glass slides. After deparaffinization in xylene, tissue sections were dehydrated in absolute ethanol and rehydrated in gradual alcohols (95–70%); sections were then rinsed three times with phosphate-buffered saline (PBS) for 5 min. Further, tissue sections were blocked in PBS containing 5% bovine serum albumin (BSA) and Tween-20 (10%). The primary rabbit polyclonal antiserum BP7 (kindly provided by Dr T.Hsu, Stanford University) against the extracellular domain of LGR7 was diluted 1:800 in antibody dilutent (5 ml PBS, 50 µl Tween 20 at 10% and 25 mg bovine serum albumin). Sections were incubated for 2 h at 37°C in a moist chamber and then washed three times for 5 min each in PBS with 10% Tween-20. Negative controls were performed in all cases by using the same protocol but in the absence of primary antibody or non-specific sera. After incubations with the primary antibody, sections were incubated with the secondary antibody for 1 h, in a moist chamber at 37°C. Sections were washed extensively in PBS with 10% Tween-20, and streptavidin peroxidase was added (Dako LSAB+ Kit). Color reaction was performed by using a 3,3'-diaminobenzidine chromogen solution, counterstained with haematoxylin and mounted with Eukitt mounting medium (O.Kindler GMBH and Co.). Examination was performed under brightfield microscope (Nikon, USA).
Cell culture
We have used THP1 cells as positive control for LGR7 mRNA and protein localization. For immunohistochemistry, THP1 cells were methanol:acetic acid (3:1) fixed and mounted on glass slides before undergoing the standard immunohistochemical protocol. This cell line is derived from the peripheral blood of a 1 year old male with acute monocytic leukaemia and it has been characterized by possessing relaxin receptors. Recently, this cell line has been used for signalling pathways studies of LGR7 (Bartsch et al., 2001).
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LGR7 mRNA expression during the menstrual cycle
LGR7 expression in human endometrial samples throughout the natural menstrual cycle was initially analysed by semi-quantitative RT–PCR. A band of the expected size of 300 bp was obtained in all samples (Figure 1).
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Sequence analyses confirmed the presence of LGR7 sequence. Subsequently, real time quantitative RT–PCR was performed on 13 endometrial samples throughout the menstrual cycle; early–mid-proliferative phase (n = 2), late proliferative phase (n = 3), early secretory phase (n = 2), mid-secretory phase (n = 3) and late secretory phase (n = 3). Maximal LGR7 mRNA expression was observed during the proliferative phase (Figure 2). However, large individual variation in the level of LGR7 expression was observed, making it impossible to determine a significant regulation in LGR7 expression. As a trend, LGR7 expression decreased after the beginning of the secretory phase and remained low, with the exception of one patient in the late secretory phase group.
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Relaxin is hypothesized to play a role during early embryonic implantation and decidualization. Therefore we analysed the expression of LGR7 in the early luteal phase in more detail. To this end, endometrial biopsies ranging from 1 to 7 days after the LH surge were collected. Q-PCR on these samples showed again a substantial individual variation in LGR7 expression. However, no consistent regulation in LGR7 expression was observed during this time span (Figure 3).
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We reasoned that because of the large individual variation in LGR7 gene expression, potential regulation during the formation of a receptive endometrium might be observed only within the same individual, preferably in the same menstrual cycle. Therefore, endometrial biopsies were collected 2 days (non-receptive) and 7 days (receptive endometrium) after the LH surge in the same individual. In these ‘coupled’ biopsies, LGR7 expression was analysed and regulation between the non-receptive and receptive endometria was determined for each patient. In five out of 13 patients LGR7 expression increased significantly (>2-fold) in time, whereas in two patients LGR7 decreased >2-fold. In the remaining six patients, LGR7 expression was not significantly regulated (Figure 4). These results indicate that there is no coordinate regulation of LGR7 expression during the formation of a receptive endometrium.
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LGR7 protein localization during the menstrual cycle
To analyse LGR7 protein localization, at least three endometrial samples per group of the menstrual cycle were immunohistochemically analysed using the BP7 antibody. For negative controls, the absence of the primary antibody or non-specific sera were used and for positive controls THP1 cells were employed. Specific endometrial LGR7 staining was observed in all phases of the menstrual cycle (Figure 5). More intense staining was observed during the proliferative phase compared to the secretory phase. In the early–mid-proliferative phase, when the endometrium is under the influence of estradiol, LGR7 staining is present at both the stromal and the epithelial compartment at the plasma membrane and cytoplasm (Figure 5A). This immunoreactive pattern was maintained in the late proliferative and early secretory phase (Figure 5B and C). However, during the mid-secretory phase, LGR7 immunoreactive protein disappeared from the stromal compartment and staining was restricted in the glandular and the luminal epithelium to the apical membrane. (Figure 5D, E and F). During the late secretory phase, staining reappeared in both stromal and epithelial compartments at the plasma membrane and cytoplasm (Figure 5G).
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The relaxin field has struggled for decades with an inability to clone a high affinity relaxin receptor. The finding by Hsu et al. (2002) that the GPCR LGR7 and LGR8 represent the receptors for relaxin, certainly came as a surprise and provided an important impulse to many areas of research where a role for relaxin has been described or hypothesized. One of these areas is uterine physiology and embryonic implantation.
As early as the 1960s it was shown that crude preparations of relaxin stimulate angiogenesis in the endometrium of immature or castrated macaques (Dallenbach-Hellwig et al., 1966; Hisaw et al., 1967). More recently it was reported that relaxin binds with high affinity to human endometrial cells (Osheroff and King, 1995), and increases VEGF secretion in a dose-dependent manner (Unemori et al., 1999). Taken together with additional data describing regulation of relaxin plasma levels during the menstrual cycle with highest levels during the late luteal phase (Stewart et al., 1990; Einspanier et al., 1999), a role for relaxin, and thus by definition also for its receptor, has been suggested in human endometrial receptivity.
The current study describes for the first time expression of LGR7 in human endometrium throughout the different phases of the menstrual cycle. The data obtained with quantitative RT–PCR clearly show that there is a large degree of individual variability in LGR7 expression and consistent regulation in the human menstrual cycle could not be demonstrated. We have chosen to study endometrium from multiple individuals taken from clearly distinguishable phases (early and mid–late proliferative; early, mid- and late secretory). The lack of clear regulation was confirmed using additional biopsies taken on well-defined time-points after the LH surge (LH+1 to LH+7) and histologically comfirmed. In an attempt to tackle the problem of individual, and possible inter-cycle, variability we evaluated endometrial samples obtained 2 and 7 days after the LH surge in the same menstrual cycle of individual patients. In total, 13 patients were analysed but no consistent regulation in LGR7 expression was observed; so no evidence was found for LGR7 transcriptional regulation. In order to extend our studies to the protein level, immunohistochemical data using an LGR7-specific antibody were obtained. We find clear staining in all phases throughout the menstrual cycle. These results are in line with the conclusions based on the Q-PCR experiments. There may be a tendency towards less staining intensity during the mid–luteal phase in which LGR7 expression disappeared from the stromal compartment but the differences are small.
Our study establishes the presence of immunoreactive LGR7 protein in the human endometrium during all phases of the menstrual cycle, with a trend to decline during the window of implantation. There are two possible explanations for the observation that immunoreactive LGR7 diminished during the mid-secretory phase, but reappeared during the late secretory phase. First, the LGR7 receptor may be occupied by its ligand during the mid-secretory phase of the menstrual cycle and therefore the occupation of the epitope prevents the antisera from binding to it. Stewart et al. (1990) showed that in non-pregnant humans the level of relaxin in serum peaks during the mid–late luteal phase. Presumably this relaxin is derived from the corpus luteum. However, there are also data supporting local production of relaxin (as a paracrine factor) within the uterus and in cultured endometrial cells, albeit at relatively low levels (Palejwala et al., 2002). At the protein level, in the marmoset monkey immunohistochemical staining for relaxin has been reported, particularly during the secretory phase of the cycle (Ivell and Einspanier, 2002; Einspanier et al., 1997). The second possibility is that progesterone down-regulates LGR7 in the human endometrium, and this is in line with the quantitative PCR results shown in Figure 2. Since it ‘takes two to tango’, it is also important to understand local relaxin concentrations in the human endometrium during the menstrual cycle.
When interpreting data related to relaxin physiology, it should be emphasized that relaxin is one of the members of a much larger family of peptide hormones, many of which have not yet been functionally characterized. It has already been hypothesized that this may lead to compensatory mechanisms, which may explain the lack of an implantation-related phenotype in relaxin knockout mice (Zhao et al., 1996). A role for relaxin family members, which may be present either in the circulation or locally, cannot definitely be excluded, even though they may have a much lower affinity for LGR7 as compared to relaxin.
LGR8 has also been reported to be specifically activated by relaxin, albeit at much higher concentrations. However, a role for LGR8 in the mediation of relaxin effects in the endometrium seems unlikely since we have not been able to detect LGR8 mRNA in endometrial biopsies (data not shown).
The data described provide the first evidence for LGR7 expression in human endometrium during the menstrual cycle and no clear evidence was obtained for its hormonal regulation. However, the large degree of variability in individual endometrial samples may mask LGR7 expression regulation. Although the mRNA data are in line with the immunohistochemical results, larger numbers of samples need to be investigated.
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Acknowledgements |
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We would like to express our gratitude to Dr Teddy Hsu from Stanford University who generously provided us with the polyclonal rabbit anti-human antiserum BP7, Dr Esperanza Gómez from the Universidad Complutense of Madrid who provided THP-1 cells, and Dr Paloma Sanz of La Princesa Hospital of Madrid who donated the liver RNA. We also thank Julio Martin and the IVI Foundation group for their technical support. This investigation has been supported by Grant SAF 2001-2948 from Ministerio de Ciencia y Tecnología from the Spanish Government.
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Submitted on September 24, 2003; accepted on November 12, 2003.
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