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Corticotropin-Releasing Hormone and Proopiomelanocortin-Derived Peptides Are Present in Human Myometrium

Departments of Physiology and Obstetrics and Gynecology (V.L.C., T.G.T., S.J.L., J.R.G.C.), University of Toronto, Toronto, Ontario, Canada M5S 1A8; Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Ontario, Canada, M5G 1X5; and the Medical Research Council Group in Fetal and Neonatal Health and Development, the Medical Research Council Group in Development and Fetal Health, and the Department of Obstetrics and Gynecology (J.F.T., A.J.T., I.T.C.), University of Glasgow, Glasgow, Scotland G3 8SJ

Address all correspondence and requests for reprints to: Dr. John Challis, Department of Physiology, University of Toronto, 1 Kings College Circle, Toronto, Ontario, Canada M5S 1A8.

	Abstract

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CRH and POMC-derived peptides are produced at a number of intrauterine sites in both the nonpregnant and pregnant states. It is hypothesized that CRH and POMC-derived peptides may be produced locally by the uterus to modulate myometrial contractility. This study has examined the distribution of these peptides in human uterine tissue during the ovulatory cycle and pregnancy. The immunoperoxidase staining method was used to localize CRH and POMC-derived peptides: ACTH, ß-endorphin, and MSH. Immunoreactive (IR-) CRH and IR-POMC-derived peptides, ß-endorphin and MSH, were observed in the myometrial smooth muscle, vascular smooth muscle, endometrial glandular epithelium, and luminal epithelium of the nonpregnant uterus (n = 17). Staining for IR-CRH did not change during the cycle from the proliferative (n = 8) to the secretory phases (n = 9). Conversely, staining for IR-ß-endorphin and IR-MSH was only observed during the secretory phase of the cycle (n = 9). In uterine tissue obtained from pregnant women (n = 20) IR-CRH was present in the myometrial smooth muscle, vascular smooth muscle, decidua, and glandular epithelium. IR-POMC-derived peptides were not detectable at any uterine site during pregnancy (n = 20). IR-CRH was measurable in myometrial extracts collected from pregnant women undergoing cesarean section (20.9 ± 3.8 ng/g wet wt; n = 7) and from nonpregnant premenopausal women undergoing hysterectomy (7.7 ± 2.1 ng/g wet wt; n = 6). IR-CRH concentrations significantly increased with pregnancy. Levels of messenger ribonucleic acid encoding for CRH were examined in nonpregnant (n = 4) and pregnant (n = 10) myometrial smooth muscle and were also significantly increased with pregnancy. This study has demonstrated that levels of CRH and POMC peptide in human uterine tissue change with pregnancy and that CRH is produced locally by myometrial smooth muscle cells. These studies are consistent with the possibility that the CRH peptide has an autocrine/paracrine activity during pregnancy and labor that may be related to the modulation of myometrial contractility.

	Introduction

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CRH, A 41-amino acid neuropeptide, is produced at a number of intrauterine sites during pregnancy, including the placental syncytiotrophoblast, amnion, chorion, and decidua (1). In nonpregnant women, CRH peptide and messenger ribonucleic acid (mRNA) have been identified in the endometrial glandular epithelium and ovary (2, 3, 4). CRH acts on the pituitary to stimulate POMC-derived peptide release and may play a similar role in uterine physiology. Endometrial cells express the POMC gene and produce its end product, ß-endorphin (4), suggesting that CRH may play a paracrine role in the nonpregnant uterus. In the human myometrium during pregnancy, it is proposed that placentally derived CRH may mediate myometrial contractility (5, 6, 7), modulate placental blood flow (8, 9), stimulate PG (10) and POMC-derived peptide production (11), and activate the fetal hypothalamic-pituitary-adrenal axis (12). As CRH is produced at some uterine sites in nonpregnant and pregnant women, it is possible that this hormone has paracrine/autocrine effects in this tissue. However, the distribution of the CRH and POMC-derived peptides, ß-endorphin, MSH, and ACTH, has not been assessed in uterine tissue from both nonpregnant and pregnant women. The present study examined the localization and distribution of CRH and POMC-derived peptides in nonpregnant and pregnant uteri. Furthermore, immunoreactive (IR-) CRH concentrations were measured in nonpregnant and pregnant myometria. CRH mRNA expression has been characterized in endometrium and decidua (2, 3, 4), but there is no evidence whether it is synthesized in the myometrium. Therefore, this study also examined changes in CRH mRNA levels in myometrial tissue collected from nonpregnant and pregnant women.

	Materials and Methods

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Nonpregnant uterine tissue was obtained from normally cycling, premenopausal women undergoing hysterectomy for benign disease at the Royal Infirmary and Western Infirmary (Glasgow, UK). Macroscopically normal tissue was sampled from the upper fundus in areas without fibroid tissue.

The nonpregnant uterine samples were dated independently by a hospital pathologist based on histological characteristics (13). Eight women were in the proliferative phase of the cycle [early proliferative (days 1–4), n = 2; midproliferative (days 5–9), n = 3; late proliferative (days 10–13), n = 3]. There were nine women identified in the secretory phase [early secretory (days 14–18), n = 3; midsecretory (days 19–24), n = 4; late secretory (days 25–28), n = 2].

Uterine tissue was obtained from women with term pregnancies during either elective cesarean section or cesarean section during labor. Tissues were collected at Mount Sinai Hospital (Toronto, Canada). Labor was defined to be of spontaneous onset; the cervix was fully effaced and at least 5 cm dilated. Cesarean sections during labor were performed for fetal distress rather than for failure to progress in labor or failed induction. In all cases a small amount of uterine tissue was removed from the upper margin of the uterine incision of the lower segment. Mean gestational age was 39.8 ± 1.2 weeks. The age range of all women in this study was 27–52 yr (n = 51), with a median age of 42 yr.

Informed consent was obtained in each case, and approval for the study was granted by the Royal Infirmary, Western Infirmary, and Mount Sinai Hospital ethics committees.

Immunohistochemistry

Tissues were formalin fixed and paraffin embedded. Sections of uterine tissue were mounted on slides coated with 3-aminopropyltriethoxy-silane (Sigma Chemical Co., St. Louis, MO). A specific rabbit antiovine antibody for CRH, three rabbit antihuman antibodies for ACTH (Serotec, Toronto, Canada; Dako Corp., Carpinteria CA; Incstar Corp., Stillwater, MN), and rabbit antihuman antibodies for ß-endorphin (Incstar Corp.) and MSH (INCSTAR Corp.) were used in conjunction with avidin-biotin-peroxidase reagents (Vectastain ABC kits, Vector Laboratories, Inc., Burlingame, CA) as previously described (14, 15).

Negative controls were conducted in which uterine sections were incubated with nonimmune rabbit serum, antibody dilution buffer alone, or primary antibody that had been preabsorbed with 1 µmol/L CRH, ACTH, ß-endorphin, or MSH (Peninsula Laboratories, Inc., Belmont, CA). Positive controls consisted of ovine fetal hypothalami that were stained for CRH and ovine fetal pituitary that were stained for POMC-derived peptides. All nonpregnant uterine experimental sections were stained simultaneously to allow direct comparison between peptides, and then all uterine samples collected from pregnant women were stained simultaneously with some nonpregnant samples as controls. All sections were examined by light microscopy and qualitatively assessed. Sections were examined by at least 2 individuals, with 10 sites examined on each section. A positive result was recognized when at least 80% of the sites examined contained positive preabsorbable staining.

CRH RIA

IR-CRH was assayed in myometrial tissue collected from nonpregnant and pregnant women, aged 25–52 yr. Myometrial tissue was sampled from the lower segment of the uterus in both groups. All decidual tissue was removed, and then the myometrial tissue was snap-frozen in liquid nitrogen. All samples were weighed and then homogenized in ice-cold deionized water. Samples were heated at 85 C for 15 min, cooled, and centrifuged at 10,000 rpm for 40 min. The supernatant was collected, lyophilized, and reconstituted in 1 mL RIA buffer (0.1 mol/L sodium phosphate, 0.25% BSA, 0.1% ß-mercaptoethanol, and phenol red, pH 7.4). Samples were then diluted and assayed for IR-CRH using an ovine CRH antibody, synthetic human CRH (Peninsula Laboratories) as standard and [¹²⁵I]Tyr-CRH, which had been purified in a propan-1-ol gradient in 1% trifluoroacetic acid over a C₁₈ Sep-Pak and then purified by high pressure liquid chromatography. The assay sensitivity was 10 pg/mL extracted myometrium, and the interassay coefficient was 21.5%.

RNA extraction

Myometrial tissue with the decidua removed was pulverized in liquid nitrogen and homogenized in 4 mol/L guanidinium isothiocyanate at room temperature. Total RNA was extracted according to the method of Chomcyznski et al. (16). Total RNA was cleaned from DNA contamination by digestion with ribonuclease-free deoxyribonuclease I.

Generation of PCR products

Complementary DNA (cDNA) from myometrial tissue was generated in a 20-µL reaction mixture containing 1 µg total RNA, 5 ng/µL random hexamers (Pharmacia Biotech, Piscataway, NJ), 100 U Moloney murine leukemia virus reverse transcriptase (Life Technologies, Gaithersburg, MD), 5 mmol/L MgCl₂ (Perkin Elmer/Cetus, Norwalk, CT), 1 x PCR buffer [10 mmol/L Tris-HCl, 50 mmol/L KCl, Perkin Elmer/Cetus), 1 mmol/L each of deoxy (d)-NTP, dATP, dCTP, dGTP, and dTTP (Pharmacia)], and 1 U/µL ribonuclease inhibitor (Boehringer Mannheim, Indianapolis, IN). To control for nonspecific amplification, the PCR mixture was also conducted in the absence of RNA (distilled water blank). This mixture was incubated at 25 C for 10 min, followed by 42 C for 30 min. The reaction was terminated by heating at 99 C for 5 min. RNA was checked for DNA contamination by performing PCR analysis on RNA, without the reverse transcriptase step, and was purified by deoxyribonuclease I digestion if positive amplification was observed.

Standard PCR was performed using 10 µL total RT mixture in a total volume of 40 µL containing 0.25 mmol/L dNTP, 50 ng of each PCR primer, and 2.5 U Taq polymerase (Boehringer Mannheim) in 1.25 mmol/L MgCl₂, 50 mmol/L KCl, and 10 mmol/L Tris-HCl (pH 8.3). The amplification profile involved preincubation at 94 C for 5 min, denaturation at 94 C for 1 min, primer annealing at temperatures specific for each primer for 1 min, and extension at 72 C for 1 min. PCR products were electrophoresed in 1.5% agarose gels, stained with ethidium bromide (Sigma Chemical Co.) in Tris-acetate-ethylenediamine tetraacetate buffer to visualize amplification products.

Semiquantitative PCR

Semiquantitative analysis provides a means of analyzing the differential expression of genes. A linear range of expression of the gene is identified through different PCR cycles, and the mean values of gene expression can be used to compare different samples. The cDNA sequence of CRH was amplified in a standard PCR reaction as described above. The following primers were used: 5'-GTGGAGAAACTCAGAGACCA-3' (corresponding to nucleotide positions 329–349) and 5'-TGTTGCTGCCGCAGCTGCT-3' (corresponding to nucleotide positions 1659–1677). An intron from nucleotide position 504-1304 was subtracted from the first and second primers so that the size of the target area of amplification was 550 bp. The amplification profile involved preincubation at 95 C for 1 min, followed by PCR cycles each with denaturation at 94 C for 1 min, primer annealing at 52 C for 1 min, and extension at 72 C for 1 min. Initially, PCR cycles were performed for 25–35 cycles.

To quantify PCR reactions, negatives were made of the PCR gels to facilitate the reading of relative optical densities of the PCR products. Relative optical density values were graphed to obtain linearity, and the optimal PCR cycles established for CRH were 23, 25, and 27 cycles.

Myometrial CRH gene expression was related to myometrial smooth muscle cell content using a gene for calponin, a cytoskeletal protein that is specific for smooth muscle cells (17).

The primers used for calponin expression were 5'-GATGGCATCATTCTTTGCGA-3' (corresponding to nucleotide positions 240–259) and 5'-TTGTAGTAGTTGTGTGCGTG-3' (corresponding to nucleotide positions 939–958). The target area of amplification was 718 bp. The specific annealing temperature was 53 C, and the PCR cycles used to determine linear expression were 17, 19, and 21 cycles.

Statistical analysis

All values were expressed as the mean ± SEM. IR-CRH levels were analyzed using Student’s t test. Differences in CRH gene expression were analyzed using the Wilcoxon-Mann-Whitney nonparametric test, and statistical significance was represented by P < 0.05.

	Results

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IR-CRH was localized in the endometrial glandular epithelium, luminal epithelium, vascular smooth muscle, and myometrial smooth muscle of the nonpregnant uterus (n = 17; Fig. 1, a and b, and Table 1). IR-CRH was present in the endometrial glandular epithelium and myometrial smooth muscle during the proliferative and secretory phases of the ovulatory cycle. Preabsorption of CRH antibodies with excess CRH peptide (1 µmol/L) abolished peroxidase staining of uterine tissue (Fig. 1, c and d). IR-CRH staining was localized to the cytoplasm of the epithelium and smooth muscle cells.

View larger version (130K): [in this window] [in a new window]   Figure 1. Localization of IR-CRH in the human nonpregnant uterus during the proliferative and secretory phases of the ovulatory cycle. Positive staining for CRH was observed in the endometrial glandular epithelium (g), myometrial smooth muscle (m), and luminal epithelium (e) in both the proliferative and secretory (A and B) phases. Preabsorption completely abolished positive staining for IR-CRH at these sites (C and D). Photographs are at x200 magnification, and the scale bar represents 90 µm.

In uterine tissue collected from pregnant women, positive staining for IR-CRH was observed in the decidual stromal cells, glandular epithelium, myometrial smooth muscle, and vascular smooth muscle (Table 2; n = 20). IR-CRH in myometrial smooth muscle decreased with labor, as only 4 of the 10 uterine samples obtained during labor were CRH positive (Table 2 and Fig. 2e), whereas all 10 of the samples collected before the initiation of labor were CRH positive (Fig. 2c and Table 2). Both the before and during labor groups were positive for IR-CRH in the decidual tissue (Table 2). IR-CRH staining in vascular smooth muscle was only observed in samples collected during labor (Table 2). The POMC-derived peptides, ACTH, ß-endorphin, and MSH, were not detectable at any uterine site before or during labor (n = 20; data not shown). All POMC-derived peptides were detectable in fetal sheep pituitary.

View larger version (185K): [in this window] [in a new window]   Figure 2. Distribution of IR-CRH in myometrial smooth muscle and glandular epithelium of nonpregnant and pregnant uterus. a, IR-CRH was present in the myometrial smooth muscle (m) and glandular epithelium (g) of nonpregnant uterus. c, IR-CRH was present in myometrial smooth muscle and glandular epithelium before the initiation of labor. e, The intensity of staining for IR-CRH decreased in the myometrial smooth muscle, but did not change in the glandular epithelium of pregnant uterus collected during labor. Preabsorption abolished positive staining for IR-CRH at these sites (b, d, and f). Photographs are at x200 magnification, and the scale bar represents 90 µm.

View larger version (11K): [in this window] [in a new window]   Figure 3. IR-CRH in pregnant and nonpregnant myometria. Myometrial samples were collected from pregnant women undergoing cesarean section (n = 7) and from women undergoing hysterectomy (n = 6). Samples were extracted in heated dH2O and assayed for CRH using RIA. Final IR-CRH concentrations were expressed as nanograms per g wet wt of myometrium. There was a significant increase in IR-CRH concentrations in pregnant myometrium compared to those in the nonpregnant state (P < 0.05, by Student’s t test).

View larger version (44K): [in this window] [in a new window]   Figure 4. CRH gene expression in human myometrial smooth muscle cells. Semiquantitative PCR studies were conducted with myometrial cDNA at an annealing temperature of 52 C and at optimal PCR cycles of 23, 25, and 27 for CRH. A 550-bp product was the target area for the CRH gene. CRH expression was standardized to the smooth muscle cell-specific gene, calponin. PCR for calponin was conducted at an annealing temperature of 53 C and at cycles 17, 19, and 21. A 719-bp product was the target area for the calponin gene.

	Discussion

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We have identified CRH mRNA in both the nonpregnant and pregnant myometrial smooth muscle, with levels of mRNA increasing during pregnancy. CRH mRNA has previously been localized in the glandular epithelium of the endometrium (4). The sizes of endometrial and decidual CRH mRNA transcripts were 1.3 and 1.2 kb, respectively, similar to hypothalamic CRH mRNA (4, 18). Petraglia et al. (18) report that CRH mRNA levels rise with gestational age and peak at term in decidualized stromal cells, and this pattern is similar to our findings in the myometrium. However, this trend may only be applicable to the lower segment of pregnant uterine tissue.

CRH may have paracrine/autocrine actions in the human uterus, as the CRH receptor is detectable in both pregnant and nonpregnant uteri (19, 20, 21, 22). The human myometrium contains a 70-kDa CRH receptor that has increased affinity for CRH at term (19). The CRH receptor was shown to be linked to adenylate cyclase and/or cyclooxygenase (20). The functional capacity of the CRH-receptor complex to activate cAMP production appears to be decreased at term, possibly allowing for activation of contractile pathways (21). Isoelectric focusing experiments have identified five isoforms of the CRH receptor in human pregnant and nonpregnant uteri, suggesting that CRH may have multiple roles in this organ (22).

The role of CRH in the human uterus remains to be determined. CRH potentiates oxytocin- and PGF₂-induced myometrial contractility in vitro (5, 7), and in vivo studies have found a positive association between maternal plasma CRH concentrations and uterine contractility in women who entered labor by receiving infused oxytocin (6). Those subjects with high CRH concentrations spent a greater proportion of time contracting and had shorter labors than those women with low plasma CRH levels. The localization of CRH in myometrial smooth muscle cells suggests that CRH may be produced locally to modulate myometrial contraction. In the nonpregnant uterus, CRH may modulate myometrial contractility for ovum transport along the Fallopian tube and sperm transport.

CRH was observed in vascular smooth muscle cells of the uterus, suggesting that it may be involved in the modulation of uterine vascular tone. Clifton et al. (8, 9) demonstrated that CRH was a potent vasodilator in the fetal-placental circulation via nitric oxide (NO). Constitutive NO synthase, the enzyme that converts L-arginine to L-citrulline and NO, has been identified in human myometrium and endometrium (23, 24). CRH may induce uterine vascular relaxation through the NO pathway.

It can be concluded from this study that CRH and POMC-derived peptides are produced in the nonpregnant uterus, consistent with these peptides playing an important role in uterine function and/or activity. The rise in CRH peptide and mRNA levels with pregnancy is consistent with the possibility that CRH may have paracrine/autocrine effects on myometrial contractility in the lower segment of the uterus.

Received June 27, 1997.

Revised June 1, 1998.

Accepted June 30, 1998.

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