Hum. Reprod. Advance Access originally published online on August 18, 2006
Human Reproduction 2007 22(1):92-96; doi:10.1093/humrep/del331
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Secretory endometrium highly expresses urocortin messenger RNA and peptide: possible role in the decidualization process
1 Department of Paediatrics, Obstetrics and Reproductive Medicine 2 Department of Human Pathology and Oncology, University of Siena, Siena and 3 Department of Obstetrics and Gynaecology, University of Milan, Milan, Italy
4 To whom correspondence should be addressed at: Chair of Obstetrics and Gynaecology, Department of Paediatrics, Obstetrics and Reproductive Medicine, University of Siena, Policlinico "Le Scotte" Viale Bracci, 53100 Siena, Italy. E-mail: petraglia{at}unisi.it
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Abstract |
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BACKGROUND: Urocortin (UCN) gene expression and synthesis have been reported in epithelial and stromal cells of the human endometrium. In this study we evaluated (i) UCN messenger RNA (mRNA) expression and peptide production in uterine specimens collected throughout the endometrial cycle, (ii) UCN secretion after decidualization of cultured human endometrial stromal cells (HESCs) and (iii) the effect of UCN on endometrial decidualization. METHODS: HESCs were isolated from samples of human endometrium collected from healthy patients with normal menstrual cycle and cultured in presence of cAMP, 17-beta-estradiol (E2) + medroxyprogesterone acetate (MPA) and UCN. UCN levels were measured in endometrial extracts by an enzyme immunoassay, and changes of endometrial UCN mRNA expression were measured by RT–PCR analysis. RESULTS: UCN peptide concentrations and mRNA expression were highest in the secretory phase of the menstrual cycle (P < 0.001, late secretory versus early and late proliferative phase) and higher in the late than the early secretory phase (P < 0.01). After decidualization of HESC with cAMP or E2 + MPA, UCN levels rose in parallel with prolactin concentrations by days 6 (P < 0.01, for all). Finally, the addition of UCN to HESCs, with or without E2 + MPA, induced the release of prolactin. CONCLUSIONS: The evidence that (i) UCN is highly expressed in the secretory phase of the endometrial cycle; (ii) cAMP and E2 + MPA modulate secretion of UCN and (iii) UCN induces HESCs decidualization together suggest a possible role for UCN in endometrial physiology.
Key words: corticotrophin-releasing factor receptor/decidualization/endometrium/urocortin
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Introduction |
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The human endometrium undergoes morphological and functional changes during the menstrual cycle to prepare the local environment for the blastocyst implantation and for the fine tunnelling of trophoblast invasion when pregnancy is achieved. Decidualization of the human endometrium is an essential preparative event to enable and to regulate embryo placentation, and it involves differentiation of stromal cells, through tissue remodelling and an inflammatory-like response. Although the exact molecular pathways involved in decidualization still remain to be elucidated, progesterone is the major physiological factor that drives both the endometrial differentiation and the local secretion of several hormones.
Urocortin (UCN) is a 40-amino acid neuropeptide that shares 45% sequence homology with corticotrophin-releasing factor (CRF) (Vaughan et al., 1995
) and which, like CRF, acts in vitro to release adrenocorticotrophic hormone from dispersed rat anterior pituitary cells (Asaba et al., 1998). The homology with CRF is also underlined by the fact that UCN binds with different affinity to both CRF receptors type 1 (CRF-R1) and 2 (CRF-R2) (Aguilera et al., 2004). UCN gene is expressed by epithelial and stromal cells of the human endometrium, and the peptide is localized in the endometrial luminal and glandular epithelial cells, and in stromal cells of both proliferative and secretory endometrium (Florio et al., 2002). Moreover, the human endometrium also expresses both CRF-R1 (Di Blasio et al., 1997) and CRF-R2 (Karteris et al., 2004), and in vitro studies have shown that CRF binding to CRF-R1 on endometrial stromal cells activates the cAMP pathway and endometrial differentiation (Ferrari et al., 1995). Owing to the biochemical (sequence homology) and biological (activation of CRF receptors) similarity between CRF and UCN, and in view of the fact that CRF has important roles in endometrium (Florio et al., 2004), in this study we investigated: (i) the changes of UCN messenger RNA (mRNA) and peptide expression in samples of endometrium collected throughout the menstrual cycle, (ii) the secretion of UCN from cultured human endometrial stromal cells (HESCs) after decidualization induced by estrogen and progesterone and (iii) the impact of UCN treatment in vitro on HESC decidualization.
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Materials and methods |
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Endometrial samples collection
Samples of endometrium were obtained from healthy fertile patients (n = 35; age range 20–31 years) with normal ovulatory menstrual cycles, who underwent hysteroscopy for evaluation of the morphology of the uterine cavity. Informed consent was obtained from all patients before inclusion in the study, for which local Human Investigation Committee approval was obtained. A complete medical history was obtained, and a physical examination was performed for each subject. Patients with pituitary, thyroid or adrenal disorders were excluded from the study. None of the subjects had taken any hormones for at least 6 months before the study, and all participants had never used hormonal preparations. The stage of the menstrual cycle [n = 8 early (EP; days 4–7) and n = 8 late (LP; days 12–15) proliferative; n = 9 early (ES; days 16 –19) and n = 10 late (LS; days 23–27) secretory endometrial samples] was initially determined from the number of days since the last menstrual period and confirmed by transvaginal ultrasound scans (Real Time Ultrasound Scan Equipment, Siemens Sonoline ELEGRA® Millenium Edition, with a transvaginal probe at 4.5–7.0 MHz) (Severi et al., 2003
) and by the histological criteria of Noyes et al. (1975). Immediately after collection, an aliquot of samples was frozen and stored at –80°C until RNA extraction.
Endometrial extracts preparation
Endometrial samples were thawed and weighed. They were then placed in 1 ml of 0.1 M acetic acid in polypropylene tubes and boiled for 10 min. After cooling on ice, the samples were homogenized (Polytron, Kinematica, Kriens-Lucerne, Switzerland), and the homogenates were centrifuged at 13000 x g for 15 min at 4°C. The supernatants were collected and stored at –80°C until UCN assay.
RNA isolation
Total RNA was extracted from frozen tissue samples using a commercially available kit (Trizol; Invitrogen, Milan, Italy). Approximately 5µg of total RNA was subsequently treated with DNase (DNase I Set; Promega, Milan, Italy). Quantification of total RNA was performed by measuring the absorbance at OD260. The quality of total RNA was checked by running 1.5% agarose gels buffered in 89 mM Tris, 89 mM boric acid and 2 mM EDTA (pH8.3) and assessed as acceptable if strong and intact 28S rRNA and 18S rRNA bands were visible under UV light after staining with ethidium bromide. No bands of genomic DNA were observed in agarose gels after DNase treatment. cDNA synthesis from total RNA (1µg) was carried out in a reaction volume of 20µl containing 50 mM Tris–HCl (pH8·3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 5µM random hexamer primer, 2·7 mM deoxynucleoside triphosphate (all reagents obtained from Invitrogen) and Moloney murine leukaemia virus reverse transcriptase (Ambion, Celbio SpA, Pero, Italy) in the presence of RNAsin (Ambion). RNA was initially denatured at 85°C for 5 min. The reaction mixture was then added, and reverse transcription was performed at 42°C for 90 min. The reaction was stopped by denaturing the enzyme at 85°C for 15 min. The cDNA was immediately subjected to qualitative PCR and quantitative real-time RT–PCR. For each RNA sample, a parallel reaction tube was prepared as described above, but without reverse transcriptase (RT-negative control).
Real-time quantitative RT–PCR analysis
To quantify changes of UCN mRNA expression, real-time quantitative RT–PCR was performed by using SYBR® Green I dye kit [based on a uniquely modified Thermus brockianus (Tbr) DNA polymerase], according to the manufacturer’s instructions (Finnzymes, Espoo, Finland), using a DNA Engine Opticon 2 (MJ Research, Bio-Rad Laboratories, Waltham, MA, USA). All samples were run in duplicates in 96-well optical PCR plates (Applied Biosystems, Weiterstadt, Germany), and standard RNA preparations were included in every RT–PCR run.
UCN was normalized to hypoxanthine phosphoribosyltransferase (HPRT; used as a housekeeping gene). The specific primers used to amplify cDNA fragments corresponding to UCN (Gene bank access no. NM003353) and HPRT (Gene bank access no. NM000194) were 5'-GCTTGCTGGTGAAAAGGACC-3' (sense) and 5'-CTTGCCCAC CGAGTCGAAT-3' (antisense) for UCN (expected size: 145bp) and 5'-TGAAGCTGCAGACACTCAGG-3' (sense) and 5'-CTCTCCC AACACCATCACCT-3' (antisense) for HPRT (included intron size: 270; expected size: 99bp). Computer analysis performed to compare the synthesized oligomers with the human sequences in the gene database of the National Center Biotechnology, using BLAST (Altschul et al., 1997), revealed no significant homology to all other genes. Sequence homology among the different oligomers used in this study was also avoided.
RNA extracted from each sample was used to amplify UCN and HPRT in separate PCRs, and all experiments were performed in duplicate. After an initial denaturation for 10 min at 95°C, the subsequent 40–50 cycles were performed using denaturation for 15s at 95°C, 15s primer annealing at 60°C for HPRT and 58°C for UCN, with a final extension at 72°C for 15s. The 
CT method (Livak and Schmittgen, 2001) was applied as a comparative method of quantification.
Cell preparation and cultures
HESCs were obtained from proliferative endometrial samples and separated as described previously (Ferrari et al., 1995) at the time the tissues were collected. Briefly, tissue samples were gently minced into small pieces (1–2 mm3) and incubated for 2h at 37°C in a shaking water bath in 10 ml of Ham’s F-10 containing 0.1% collagenase. Stromal and epithelial cells were then separated by differential sedimentation at unit gravity and selective plating on plastic substrate. The purity of stromal cells obtained by this method was usually >95%, as determined by immunohistochemical staining against vimentin (stromal cell marker) and cytokeratin (epithelial cell marker). Macrophages contamination of cultures was <2% as assessed by flow cytometric analysis. Stromal cells were cultured in Ham’s F-10 medium supplemented with 10% fetal calf serum (FCS) and antibiotics at 37°C in a 95% air and 5% CO2 incubator.
In vitro decidualization
HESCs were decidualized using two distinct methods, as previously described (Tang et al., 1993; Dimitriadis et al., 2005). HESC cultures from individual biopsies (n = 4) were incubated with or without cAMP (0.5 mM, Sigma) in triplicate for 6 days with media collection and replenishment every 48h. Alternatively, HESCs (n = 4) were treated with estradiol (E2) (10–8 M, Sigma) in the presence of medroxyprogesterone acetate (MPA; 10–7 M, Sigma) for 10 days. To evaluate the effect of UCN, HESCs (n = 4) were treated with either E2 (10–8 M, Sigma) plus MPA (10–7 M, Sigma) in presence of UCN (1 ng/ml) or UCN alone (1 ng/ml; n = 4) for 10 days. Media were collected for hormone assays. At the end of the experiment, cell number and viability were assessed by Trypan blue exclusion.
UCN and prolactin assay
Supernatants and conditioned media were assayed for UCN using an Enzyme Immunoassay Kit (Phoenix Pharmaceuticals Inc, Belmont, CA, USA) as per instructions of the manufacturer. The concentration range for UCN that is detectable by the kit is 0–100 ng/ml. Briefly, samples were treated with biotinylated rabbit anti-UCN (human) serum and were incubated for 2h at room temperature. After the wells were washed, streptavidin–horse-radish peroxidase was added and further incubated for 1 h at room temperature. Wells were washed, and the substrate 3,3',5,5'-tetramethylbenzidine solution was added and incubated for 1 h at room temperature. The reaction was then stopped by adding 2 N HCl, and the optical density (OD) was read (MR 600, Dynatech Corp., Chantilly, VA, USA).
Prolactin concentrations were measured in conditioned media using an immunometric assay purchased from Euro/DPC Ltd (Gwynedd, UK) following company instructions. The assay had a sensitivity of <0.5 ng/ml, with intra- and inter-assay coefficients of variation of 7 and 8%, respectively.
Statistical analysis
After confirming a normal distribution, data were summarized as mean ± SE. Between-group differences were evaluated by using the one-way analysis of variance (ANOVA) test followed by the post hoc Tukey’s test, and statistical significance was assumed when P < 0.05.
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UCN peptide concentrations in endometrial extracts
UCN peptide was detected in samples of human endometrium collected throughout the menstrual cycle. Levels were highest in endometrial specimens collected in the late secretory phase, being significantly higher than in those in the early and late proliferative (P < 0.001 for both), and the early secretory (P < 0.01) phases. In addition, UCN concentrations were significantly higher in the early secretory than early (P < 0.001) and late (P < 0.01) proliferative endometrium and in the late versus the early proliferative phase (P < 0.05) (Figure 1A).
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Expression of UCN mRNA
When evaluated by quantitative real-time RT–PCR, the expression of UCN mRNA was significantly (P < 0.0001) higher in the secretory than in proliferative endometrial samples (Figure 1B). Indeed, UCN mRNA expression was significantly higher in the late secretory-phase samples than in the early secretory (P < 0.01) or early and late (P < 0.001 for both) proliferative endometrial samples (Figure 1B).
UCN secretion from stromal cells decidualized in vitro
Conditioned media collected from HESCs decidualized with cAMP or E2 + MPA were assayed for UCN and prolactin. Following stimulation with cAMP, UCN levels rose in parallel with prolactin concentrations by day 6, sharing a 7-fold increase from baseline values (Figure 2) (P < 0.01). Furthermore, the addition of E2 + MPA induced UCN secretion from HESC, with the maximum effect by day 6 (Figure 2) (P < 0.001), and levels were lower than those measured after cAMP stimuli (P < 0.001 for all days of treatment) (Figures 2).
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Effect of UCN on stromal cell decidualization
The addition of UCN significantly (P < 0.01) increased the release of prolactin from cultured HESC at day 6 of culture, with the maximum effect by day 10 (Figure 3). Furthermore, inclusion of exogenous UCN with the decidualization stimulus (E2 + MPA) had no effect on viable cell numbers but stimulated prolactin secretion in significantly higher amounts than after the addition of UCN alone (P < 0.01 for all days of treatment) (Figure 3).
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Discussion |
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This study for the first time showed that human endometrial UCN mRNA and peptide expression change throughout the menstrual cycle, with the highest expression in the late secretory phase, suggesting that a correlation would exist between UCN and sex steroids, the critical modulator factors of endometrial differentiation. Indeed, when HESCs were cultured in presence of E2 + MPA, they secreted UCN in parallel with prolactin, a well-established marker of decidualization (Tang et al., 1993
), leading us to propose that UCN expression is high in the late secretory phase of the menstrual cycle because of a stimulatory effect of ovarian steroid hormones.
The present evidence that UCN, with or without the decidualization stimulus (E2 + MPA), triggers the release of prolactin does support a role for UCN in the endometrial decidualization process. Therefore, UCN may be included with CRF (Ferrari et al., 1995), interleukin 11 (IL-11) (Dimitriadis et al., 2005), prostaglandin E2 (PGE2) (Frank et al., 1994), activin A (Jones et al., 2002) and leukaemia inhibitory factor (Kimber, 2005) in the list of factors that contribute to the progression of stromal cell decidualization. Moreover, the fact that UCN is highly expressed in the secretory endometrium (present data) and that it binds to CRF receptors (Aguilera et al., 2004), together with the distribution of CRF receptors on the endometrium (Di Blasio et al., 1997; Karteris et al., 2004), immune cells (Suda et al., 2004), trophoblast cells (Florio et al., 2000) and blood vessels (Simoncini et al., 1999; Jain et al., 2000), lead us to hypothesize multiple possible sites of action for UCN in the endometrium. With respect to the endometrium, although the downstream effects of UCN during decidualization have not yet been explored, UCN may stimulate the expression of other factors involved in decidualization, including PGE2 (Frank et al., 1994) and matrix metalloproteinases (MMPs) (Li and Challis, 2005). Indeed, if pregnancy occurs, it is clearly important to inhibit the release of PGs, which occurs at the time of menstrual bleeding (Jabbour and Sales, 2004). In this regard, it is well established the role played by CRF PGs synthesis (Zoumakis et al., 2000), through the modulation of cyclooxygenase-2 (COX-2) activity, the enzyme that synthesizes PGs and is distributed in the endometrium (Jones et al., 1997). CRF suppresses the production of PGs from human endometrial cells in a time- and dose-dependent fashion (Zoumakis et al., 2000), probably through the inhibition of COX-2, as it occurs in human endothelial and fibroblast cells in culture (Fleisher-Berkovich and Danon, 1995). Thus, because UCN binds to CRF receptors (Aguilera et al., 2004), an effect on endometrial secretion of PGs may be hypothesized.
The induction of the extracellular matrix components is an essential step for the invasion of the trophoblast into the maternal decidua (Salamonsen et al., 2000). UCN is able to induce MMP-9 secretion by the human trophoblast (Li and Challis, 2005), and therefore, it may be proposed that at the time of implantation the endometrium expresses high levels of UCN that, in turn, may stimulate, through the binding to CRF receptors (Florio et al., 2000), the trophoblast to produce MMP-9 that consequently facilitates invasion of the maternal decidua.
With respect to the putative effect of UCN on immune cells, it is well known that during implantation, numerous macrophages are present at the implantation site, and this was originally thought to represent an immune response against the invading trophoblast (Abrahams et al., 2004). The evidence that (i) CRF-R1 and CRF-R2 are expressed in macrophages (Tsatsanis et al., 2005); (ii) UCN acts on macrophages to induce their apoptosis (Tsatsanis et al., 2005) and (iii) activation of the CRF-R1 pathway promotes blastocyst implantation and early maternal tolerance, primarily by killing activated T cells (Makrigiannakis et al., 2001), together lead us to suggest that UCN is expressed in high levels by the secretory endometrium, and it is mainly secreted after decidualization to act locally as an anti-inflammation agent that, by accelerating macrophage apoptosis, modulates the decidual response to the invading trophoblast. The evidence that endometrial UCN concentrations are significantly increased in women with spontaneous abortions (Madhappan et al., 2003) would support a link between UCN and successful implantation.
Finally, the findings that vascular endometrial cells express CRF-R2 (Simoncini et al., 1999; Jain et al., 2000), that UCN is a potent vasodilator (Mincheva-Nilsson et al., 1994; Schilling et al., 1998; Huang et al., 2002, 2003; Yao et al., 2002) and that UCN is expressed at high levels at the time of decidualization (present data) together suggest that UCN may be an additional peptide expressed by the human endometrium to regulate endometrial angiogenesis and/or the vascular endothelial tone, which are key events during implantation.
In conclusion, this study showed that (i) UCN mRNA is expressed, and UCN peptide is produced, at high levels in the secretory endometrium; (ii) factors that induce the decidualization of endometrial stromal cells in vitro also stimulate local UCN secretion and (iii) UCN induces in vitro decidualization of endometrial cells and also increases the effect of ovarian sex steroids hormones on cultured HESCs. A role for UCN in endometrial differentiation, trophoblast invasion and embryo implantation may be proposed.
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Submitted on February 1, 2006; resubmitted on May 11, 2006; resubmitted on July 12, 2006; accepted on July 17, 2006.
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