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Expression of Corticotropin-Releasing Hormone and Its R1 receptor in Human Endometrial Stromal Cells

Centro Auxologico Italiano, (A.M.D., F.P.G.), Milano; Department of Obstetrics/Gynecology (P.V., M.V.), 2^nd Chair of Endocrinology (F.C.), University of Milano; Department of Obstetrics/Gynecology (F.P.), University of Pisa, Italy

Address all correspondence and requests for reprints to: Anna Maria Di Blasio, MD, Centro Auxologico Italiano, Viale Montenero 32, Milan 20135, Italy.

	Abstract

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Corticotropin-releasing hormone (CRH) is a hypothalamic neuropeptide that has been identified also in several peripheral tissues, including organs of the reproductive system. In man, CRH is synthesized and released by the gonads, the placenta, maternal decidua, and the epithelial endometrium. So far, CRH has been demonstrated in endometrial stromal cells only after decidualization. The aim of this study was to seek evidence of the production and secretion of CRH by endometrial stromal cells in different phases of the menstrual cycle and to look for gene expression of the recently identified CRH receptor R1. Total RNA was extracted from stromal cells monolayers established from endometrial samples collected during both proliferative and secretive phases. After reverse transcription, polymerase chain reaction (PCR) amplification was carried out using primers specific to CRH and to CRH receptor R1, resulting in the expected bands, respectively 233 bp for CRH and 274 bp for CRH-R1. The identity of the obtained CRH PCR product was confirmed by restriction enzyme analysis and by Southern blotting. Purification by high performance liquid chromatography (HPLC) of stromal cell culture medium revealed a major peak of CRH immunoreactivity coeluting with the standard CRH(1–41), thus indicating the secretion of the mature peptide.

Our study demonstrates the synthesis and secretion of CRH by endometrial stromal cells at all phases of the menstrual cycle. We also demonstrate the expression of the CRH receptor R1 gene. It can be hypothesized that CRH contributes via autocrine/paracrine mechanisms to endometrial physiology.

	Introduction

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CORTICOTROPIN-RELEASING hormone (CRH), a 41-amino acid peptide originally isolated from the hypothalamus, is the major hypophysiotropic factor stimulating the release of ACTH and other POMC-related peptides (1). Recently, CRH and its receptors have been identified in many extrahypothalamic sites of the central nervous system (2, 3) and in several peripheral tissues (4, 5, 6). In the human reproductive system, placenta, fetal membranes, and maternal decidua synthesize and release CRH both in maternal circulation and in amniotic fluid, and CRH levels rise progressively throughout gestation (7, 8, 9). In nonpregnant uterus, CRH has been detected, both in vivo and in vitro, in normal epithelial cells and in the Ishikawa cell line, a human endometrial epithelium-derived tumor line (10). Immunoreactive CRH (ir-CRH) extracted from endometrial tissue or endometrial cells in culture has the same chromatographic profile as synthetic CRH(1–41) and is present throughout the menstrual cycle (10, 11). Moreover, as assessed by Northern hybridization, the CRH gene is expressed in endometrial epithelial cells and in their neoplastic counterparts (10).

In addition to these findings, ir-CRH has been detected in endometrial stromal cells only in the late secretory phase, when these cells undergo decidualization (11). Positive immunostaining for CRH was also observed in endometrial stromal cells decidualized in vitro after exposure to estradiol, medroxiprogesterone acetate, and relaxin (12).

The aim of this study was to investigate whether CRH is produced by cultured human endometrial stromal cells throughout the menstrual cycle. To this purpose, CRH gene expression was evaluated using reverse transcriptase-polymerase chain reaction (RT-PCR), and CRH peptide secretion was studied by high pressure liquid chromatography (HPLC). We also evaluated the expression of the gene for the newly isolated CRH receptor R1.

	Materials and Methods

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Cell preparation and culture

Endometrium was collected from eight women scheduled for laparoscopy because of infertility or pelvic pain, after informed consent was given. Laparoscopic examination demonstrated no uterine pathologies. Four specimens of endometrium were obtained in the proliferative phase and four in the secretory phase, based on the date of the last menstrual period and the histologic examination of the samples. In a previous study, we had successfully established and employed stromal and epithelial cell monolayer from normal endometrial samples (13). With this technique, diffuse and strong cytoplasmatic immunostaining for vimentin and cytokeratin is present in nearly all cultured stromal and epithelial cells, respectively. Furthermore, cytofluorimetric analysis indicates that macrophage contamination of our cultures is less than 2%. Briefly, endometrial tissue, collected at both phases of the menstrual cycle, was gently minced into small pieces (1–2 mm³/L³) and incubated for 2 h at 37 C in a shaking water bath in 10 mL Ham’s F-10 containing 0.1% collagenase. Stromal cells and epithelial glands were then separated by differential sedimentation at unity gravity and selective plating on plastic substrate. Dissociation of epithelial glands in single cells was achieved by digesting the pellet in a 0.05% trypsin solution for 3–5 min. Epithelial and stromal cells were cultured in Ham’s F-10 supplemented with 10% fetal calf serum (FCS) and antibiotics at 37 C, in a 95% air and 5% CO₂ incubator. Total RNA was extracted as described when the cultures became subconfluent. This was generally achieved after 10 days.

Reverse transcriptase-polymerase chain reaction (RT-PCR)

Total RNA was extracted from human endometrial cells according to the method of Chomczynski and Sacchi (14). The presence of CRH and its receptor messenger RNAs (mRNA) was demonstrated by amplifying the respective sequences with PCR according to the instructions provided with the GeneAmp amplification reaction kit (Perkin Elmer, Milano, Italy). One microgram of total RNA was reverse transcribed to obtain complementary (c) DNA. PCR was performed on the entire cDNA product with Taq (Thermus acquaticus) DNA polymerase and specific oligonucleotide primers.

Reaction conditions for RT were as follows: 1 mmol/L each deoxynucleoside triphosphate, 1 unit RNAsin, 100 pmol/L random hexamers, and 200 units Murine Moloney Leukemia Virus RT. The reaction was run at 42 C for 1 h. The mixture was then heated at 99 C for 5 min and quickly chilled on ice.

The primers used to amplify the sequences of the human CRH and CRH receptor R₁ cDNAs were: CRH sense 5' TTTCCGCGTGTTGCTGC 3', CRH antisense 5' TTCCTGTTGCTGTGAGC 3'; CRH R1 sense 5' GGCAGCTAGTGGTTCGGCC 3', and CRH R1 antisense 5' TCGCAGGCACCGGATGCTC 3'. Given the high homology between the rat and human CRH genes, the above mentioned primers also correctly amplify cDNA derived from rat hypothalamic RNA. Rat tissues were therefore used as negative controls. For CRH, the amplification protocol was as follows: 95 C (4 min), 56 C (2 min), and 72 C (2 min) for 1 cycle; 95 C (40 sec), 56 C (30 sec), and 72 C (1 min) for 35 cycles, followed by a 15 min extension at 72 C. For the CRH receptor cDNA amplification, conditions were: 94 C (60 sec), 63 C (60 sec), and 72 C (60 sec) for 35 cycles. DNA fragments were visualized on a 4% agarose gel stained with ethidium bromide.

In all the amplification procedures, the negative control was a blank prepared with all reagents and substituting 2 µL of water for RNA. Quantity, integrity, and possible genomic DNA contamination of all RNA samples were controlled by RT-PCR of the constitutively expressed human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as previously described (15). All GAPDH amplifications revealed the correct 240 bp fragment, but no 354 bp product that would have indicated genomic contamination.

Restriction enzyme analysis

After amplification with CRH or CRH R1 primers, PCR products were purified and concentrated using the JetPure PCR purification kit (Genomed, Bad Oeynhausen, Germany); one third of the purified DNA was incubated for 2 h at 37 C with PstI or AluI, respectively (Amersham Life Sciences, Milano, Italy). Digests were then electrophoresed and stained.

Southern blot hybridization

An aliquot of the PCR product was electrophoresed on a 4% agarose gel and transfered to a Nytran membrane (Hybond, Amersham, Milano, Italy). The membrane was prehybridized in 6 x SSC, 0.5% SDS, 5 x Denhardt’s, 0.01 mol/L Na phosphate, 1 mmol/L EDTA, and 100 mg/mL salmon sperm DNA for 2 h at 50 C. An oligoprobe complementary to nucleotides 506–530 of the human CRH gene (16) was 5' end-labeled using T4 polynucleotide kinase (New England Biolabs, Milano, Italy) and ³²P ATP (Amersham Life Sciences, Milano, Italy). The labeled oligoprobe was then added to the same solution and hybridization carried out for 4 h at 50 C. The membrane was washed three times for 2 min at room temperature (RT) and twice at 50 C in 2 x SSC, 0.1% SDS, thereafter 2 min at RT and 2 min at 50 C in 0.5 x SSC, 0.2% SDS. The filter was exposed overnight at RT to an X-ray film (X-Omat AR, Eastman Kodak, Milano, Italy).

HPLC and radioimmunoassay (RIA) for CRH

Endometrial stromal cells were incubated in serum-free medium supplemented with 0.5% bovine serum albumin for 48 h. The culture media were then collected and acidified with 1 N trifluoracetic acid (TFA) (Sigma, Milano, Italy). Samples were applied to Sep-Pack C18 columns (Millipore, Milano, Italy) previously activated with 80% acetonitrile (ACN) in 0.01 N TFA. Columns were washed with 0.01 N TFA and samples eluted with 80% ACN in 0.01 N TFA. After lyophilization, samples were reconstituted in 0.01 N TFA and applied to a Nucleosil C 18 reverse phase column (5 µm; 250 x 4.6 mm) attached to a 410 LC system (Perkin Elmer, Milano, Italy) and eluted with the following linear gradient: 0–35% ACN in 0.01 N TFA in 10 min, 35–65% ACN in 35 min, and 65–80% ACN in 5 min at 1 mL/min. One minute fractions were collected, lyophilized, and reconstituted in CRH RIA buffer (phosphate-EDTA, pH 7.4, containing 0.02% sodium azide). Following HPLC purification of endometrial stromal cell culture medium, human CRH(1–41) was loaded onto the column to determine the elution profile of the standard peptide. CRH RIA was carried out at 4 C with a 100-fold dilution of the anti-CRH antibody in normal rabbit serum and delayed addition of tracer (1000 cpm/tube). The bound fraction was precipitated by addition of a second antibody and centrifugation (5,000g for 20 min at 4 C). Assay sensitivity was 2 pg/tube, while half-maximal displacement occurred at 13 pg/tube. Intraassay coefficient of variation was 7.7%. Human CRH standard and antibodies for RIA were obtained from IgG Corporation, Nashville, TN, while ¹²⁵I-Tyro-human CRF was purchased from DuPont de Nemours, Dreieich, Germany.

	Results

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Total RNA derived from endometrial stromal cells in both proliferative and secretory phase was reverse transcribed and amplified using primers specific for the human CRH gene. Fig. 1 shows that PCR amplification generates a single DNA fragment corresponding to the expected length, 233 bp. The same amplification product can be obtained from RNA derived from endometrial epithelial cells, confirming recently published data (10). Rat liver was used as negative control, and indeed, no DNA fragment could be detected after amplification of total RNA derived from this tissue (Fig. 1). The identity of the PCR product with the primer-defined CRH sequence was demonstrated by restriction enzyme analysis and Southern blotting. The amplified sequence of the CRH gene does contain a restriction site for PstI. Consistent with this, PstI digestion of the PCR products derived from the RNA of endometrial stromal cells generates a major fragment of the expected size (Fig. 2). The CRH PCR products were also studied by Southern hybridization using an oligoprobe corresponding to nucleotides 506–530 of the human CRH gene. As indicated in Fig. 3, the fragments amplified from endometrial stromal cells in proliferative and secretory phases generated a positive hybridization signal that was not detected in the DNA fragment generated by rat liver RNA.

View larger version (114K): [in this window] [in a new window]   Figure 1. Analysis of CRH mRNA by RT-PCR in human endometrial stromal cells in both proliferative and secretory phase (lanes 1 and 2), human endometrial epithelial cells (lane 3) and rat liver (lane 4). CRH primers amplified a 233 bp fragment. Size marker is shown in the right side of the gel. Human epithelial cells and rat liver were used as positive and negative controls, respectively.

View larger version (99K): [in this window] [in a new window]   Figure 2. Restriction enzyme analysis of human CRH DNA fragments obtained from endometrial stromal cells in proliferative and secretory phase (lanes 1 and 3). Lanes 1 and 3 show the undigested PCR products, while lanes 2 and 4 show the correspondent PCR products after PstI digestion. Size marker is on the left side of the gel.

View larger version (54K): [in this window] [in a new window]   Figure 3. Southern hybridization of CRH DNA fragments derived from endometrial stromal cells in proliferative and secretory phase (lanes 1 and 2, respectively). Lane 3 shows the absence of hybridization signal in the PCR product derived from rat liver RNA.

View larger version (101K): [in this window] [in a new window]   Figure 4. Analysis of CRH receptor 1 mRNA by RT-PCR in human endometrial stromal cells in proliferative (lanes 1 and 2) and secretory (lanes 3 and 4) phase. CRH receptor primers amplified a 274 bp fragment. Lane 5 shows the DNA fragments obtained after AluI digestion (180 and 90 bp, respectively). Size marker is on the right side of the gel.

View larger version (15K): [in this window] [in a new window]   Figure 5. Reverse HPLC characterization of ir-CRH extracted from human endometrial stromal cell culture medium. The amount of ir-CRH in individual fractions was determined by RIA as described in Materials and Methods. The arrow indicates the fractions where synthetic hCRH was eluted from the column.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The presence of CRH mRNA and peptide has already been described in the epithelial cell population of the nonpregnant uterus (10). To the best of our knowledge, this is the first report that the CRH gene is also expressed in the stromal compartment of the endometrium.

Our results seem to be in contrast with previously published studies that investigated the presence of immunoreactive CRH in endometrial stromal cells both in vitro and in vivo (11, 12). These studies did not observe any positive staining for CRH in stromal cells during the proliferative and early secretory phase of the cycle. However, the CRH peptide was detectable in the late secretory phase, when decidualization of stromal cells occurred (11). The same results were obtained when endometrial stromal cells underwent decidualization in vitro following exposure to estradiol, medroxiprogesterone acetate, and relaxin (12). The contrast between our present data and these previous findings may be more apparent than real. In the present study, we evaluate CRH gene expression using RT-PCR, a technique that is very sensitive and is currently employed to detect low levels of mRNA expression. As the studied primary cultures were highly pure and there was no macrophage contamination at the time the experiments were performed, we are confident that no artifacts affect these results, and it can be stated that CRH gene expression occurs in endometrial stromal cells throughout the menstrual cycle. The positive RT-PCR for CRH mRNA, associated with the failure to demonstrate ir-CRH in endometrial stromal cells in proliferative and early secretory phase might indicate that, at these stages of the cycle, CRH mRNA levels are low, and consequently, CRH peptide production might be under the detection limits of immunohistochemistry. Subsequently, when decidualization occurs, CRH gene expression might be increased, resulting in higher protein synthesis. Indeed, CRH-negative endometrial stromal cells treated in vitro with progesterone and other hormones become positive for the presence of the peptide, and decidual levels of CRH mRNA increase progressively during gestation (12).

Alternatively, the absence of CRH immunoreactivity in endometrial stromal cells might be the result of the binding of the peptide to its binding protein, masking the antigenic sites. This possibility has already been suggested to explain the discrepancies obtained in some areas of the central nervous system, when the presence of CRH mRNA and peptide product was simultaneously investigated by immunohistochemistry and hybridization histochemical methods (17).

The role of CRH in endometrial physiology is still unclear. It has recently been established that, in vivo, the peptide has local inflammatory actions, and its immunoneutralization attenuates the inflammatory response (4). Thus it is possible that endometrial CRH participates in the inflammatory phenomena taking place in the endometrium.

Another local action of CRH could be the modulation of endometrial vascular tone. It has been shown that peripheral CRH exerts vasodilatory effects (18), and it is known that endometrial microvasculature is involved in the events leading to implantation (19).

Finally, in vitro, CRH is capable of inducing decidualization of endometrial stromal cells as indicated by their morphological changes and release of prolactin in the medium (20). More importantly, these effects are significantly augmented if the cells are coincubated with CRH and progesterone. Taking together our findings and these observations, it is tempting to speculate that CRH exerts an autocrine action on endometrial stromal cells in conjunction with progesterone and other local factors to induce their differentiation into the embryologic progenitors of the placental decidua.

In conclusion, we present clear evidence that synthesis and release of CRH occurs in human endometrial stromal cells in all phases of the menstrual cycle. Further studies will investigate the factor(s) that modulate CRH synthesis and how this peptide participates in the complex phenomena underlying the physiological changes of the human endometrium.

Received October 7, 1996.

Accepted January 30, 1997.

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