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Pregnancy |
Samuel Lunenfeld Research Institute,3 Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada
Institute of Medical Science4 and Departments of Obstetrics & Gynecology and Physiology,
5 University of Toronto, Toronto, Ontario M5S 1A8 , Canada
Department of Obstetrics and Gynecology,6 University of Alberta, Edmonton, Alberta T5H 3V9, Canada
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gene regulation, parturition, pregnancy, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Progesterone is a primary regulator of uterine quiescence during pregnancy mainly though the inhibition of CAP gene expression. Treatment of pregnant rats with progesterone prevents both the onset of labor and the increase in Cx43 and OTR gene expression in the myometrium [7, 8]. At term in the rat, progesterone levels in maternal circulation begin to decrease on Day 20 and continue to fall until the day of labor [9]. Several hormones produced in the intrauterine tissues inhibit myometrial contractions during gestation, including relaxin, corticotropin-releasing hormone, prostacyclin, and parathyroid hormone-related protein (PTHrP) (reviewed in [10]). These hormones therefore may have an important role in maintaining the quiescent state of the uterus until the day of labor. In general, the levels of these inhibitors peak during late pregnancy and act by elevating intracellular cAMP, leading to the activation of protein kinase A (PKA) and the subsequent inhibition of myometrial smooth muscle cell contraction (reviewed in [10]).
PTHrP is produced by many fetal and adult tissues and has several functions both during development and in adult tissues, including regulation of vascular tone, bone remodeling, placental calcium transport, and myometrial relaxation (reviewed in [11, 12]). The first 13 amino acids of this peptide hormone are highly conserved compared with the corresponding amino acids of parathyroid hormone (PTH). However, although the actions of PTH are systemic, PTHrP generally acts in an autocrine or paracrine manner. In rat myometrium, the levels of PTHrP mRNA increase during late gestation and are higher in gravid than in nongravid uterine horns [13]. In pregnant rats, PTHrP (fragment 1-34) had potent inhibitory effects on spontaneous contractions in the longitudinal layer of the myometrium, whereas in nonpregnant rats PTHrP (1-34) inhibited both oxytocin- and acetylcholine-stimulated uterine contractions [14–16]. In human myometrium, PTHrP (1-34) exerted a significant relaxant effect on late gestation tissues obtained before but not after the onset of labor [17], which suggests that with the onset of labor there is a removal of the ability of PTHrP to exert its relaxant effect.
Through alternative splicing and posttranslational processing, the PTHrP gene gives rise to a family of secretory products (reviewed in [18]). The amino-terminal PTHrP (fragment 1-36) binds to the same membrane receptor with kinetics equal to that of PTH. The PTH/PTHrP receptor has the ability to signal through both the PKA and protein kinase C pathways via coupling to guanine nucleotide-binding protein G(s), alpha subunit (G
s), and guanine nucleotide-binding protein G(11), alpha subunit (G11), and Gq/11, respectively, leading to downstream changes in gene expression [19, 20]. In a rat osteosarcoma cell line (ROS 17/2.8), receptor coupling to G-proteins depends on receptor density. At the highest receptor density, the PTH/PTHrP receptor was coupled to both Gs and Gq/11, at intermediate receptor densities only coupling to Gs was observed, and at the lowest receptor density no G-protein coupling was detected [20]. The PTH/PTHrP receptor is expressed in many fetal and adult tissues, including the uterus and ovaries of nonpregnant rats, although the highest levels of expression were found in the kidney [21].
Because PTHrP, through the PTH/PTHrP receptor, affects gene expression in other cell types, we investigated the ability of this peptide to alter the expression of labor-associated genes in the myometrium prior to the onset of labor. In this study, pregnant rats were treated with a human synthetic PTHrP fragment (1-34), and the expression in the myometrium of Cx43, OTR, c-fos, fra-2, and PTH/PTHrP receptor was investigated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female virgin Wistar rats (Charles River Co., St. Constant, PQ, Canada) were mated with male Wistar rats. Day 1 of gestation was designated as the day a vaginal plug was observed. Female rats were then housed individually under standard environmental conditions (12L:12D cycle) and fed Purina Rat Chow (Ralston Purina, St. Louis, MO) and water ad libitum. Under these conditions, the average time of delivery was at 1200 h on Day 23. Animals were considered in labor when at least one pup had been delivered. Pregnant rats were treated starting on Day 19 of gestation with daily (1000 h) i.p. injections of PTHrP (100 µg/kg human synthetic PTHrP fragment 1-34; Sigma Chemical Co., St. Louis, MO) or vehicle (sterile distilled water). This relatively high dose of PTHrP is in the same range as that shown to have effects on Cx43 expression in bone tissue [22]. All experiments were approved by the institutional animal care committee.
Tissue Collection
Myometrial samples were collected from vehicle- and PTHrP-treated pregnant rats on Gestational Days 20, 21, 22, and 23L (labor). Tissue was collected at 1000 h on all days, except Day 23 when the tissue was collected during labor. Animals were killed by decapitation, and blood was collected in tubes containing sodium heparin (Becton Dickinson, Franklin Lakes, NJ). Blood samples were centrifuged at 2500 x g at 4°C for 15 min; the plasma was then transferred to a fresh tube and stored at -70°C. Maternal serum progesterone concentrations were determined by RIA of 20 µl of rat plasma, using a Coat-A-Count kit (Diagnostic Products, Los Angeles, CA). Uterine horns were removed, bisected longitudinally, dissected away from both pups and placentas, and placed in ice-cold PBS. The endometrium was separated from the myometrial tissue by mechanical scraping on ice, a technique previously shown to remove the entire luminal epithelium and the majority of the uterine stroma [23]. The tissue was then flash-frozen in liquid nitrogen and stored at -70°C.
Northern Blot Analysis
Frozen tissue was crushed under liquid nitrogen using a mortar and pestle. Crushed tissue was homogenized for 1 min in Trizol (Gibco BRL, Grand Island, NY), and RNA was extracted according to the manufacturer's specifications. Purified RNA (10 µg) was then analyzed by Northern blot analysis. RNA was separated on a 1% (wt/vol) agarose (Gibco BRL) gel containing, 3.7% (vol/vol) formaldehyde (Fisher Scientific, Fair Lawn, NJ) in MOPS (3'-[N-morpholino]propanesulfonic acid; Sigma), transferred in 0.1 M sodium phosphate (NaP; Sigma) onto a nylon membrane (GeneScreen; DuPont, NEN Research Products, Boston, MA), and cross-linked by ultraviolet irradiation. Rat Cx43 (a gift from Dr David Paul, Department of Anatomy and Cell Biology, Harvard Medical School, Boston, MA), rat c-fos (provided by Dr. Curran, Roche Research Center, Nutley, NJ), fra-2 (GenBank U18913), rat PTH/PTHrP receptor (generated by reverse transcription polymerase chain reaction amplification of mRNA extracted from laboring myometrium using the following primers: PTHrP-R-U: 5'-GCA CGC GCA ACT ACA TCC ACA-3', PTHrP-R-L: 5'-GAC ACC AAA GAG CGG CAC GAG-3'), and 18S ribosomal protein (a gift from Dr. Denhardt, Rutgers University, Piscataway, NJ) cDNA probes were labeled with [-32P]dCTP (NEN Research Products) using the Megaprime DNA labeling system (Amersham Pharmacia Biotech UK, Little Chalfont, U.K.). Hybridization was conducted at 55°C in 30% formamide for 20 h according to the method described in Bio-Rad bulletin 1110 (Bio-Rad Laboratories, Richmond, CA). Subsequently, the membrane was washed to a final stringency of 30 mM NaP/0.1% SDS (EM Science, Darmstadt, Germany). All RNA isolation and analysis was carried out in diethyl pyrocarbonate (Sigma)-treated water. Probed membranes were exposed to x-ray film (X-Omat Blue; Eastman Kodak, Rochester, NY) with an intensifying screen at -70°C and analyzed by densitometry.
Western Blot Analysis
Myometrial tissue was pulverized under liquid nitrogen and homogenized for 1 min on ice in RIPA lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% (vol/vol) Triton X-100, 1% (vol/vol) sodium deoxycholate, and 0.1% (wt/vol) SDS supplemented with 100 µM sodium orthovanadate and protease inhibitor cocktail tablets (CompleteTM Mini; Roche, Mannheim, Germany). Samples were centrifuged at 12 000 x g for 15 min at 4°C, and the supernatant was transferred to a fresh tube to obtain a crude protein lysate. Protein concentrations were determined using the BioRad protein assay buffer (BioRad, Hercules, CA). Protein samples (50 µg) were suspended in Laemmli buffer, boiled at 95°C for 5 min, resolved by electrophoresis on a 10% SDS-polyacrylamide gel, and transferred onto polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). PVDF membranes were probed with 0.25 µg/ml affinity-purified rabbit polyclonal anti-Cx43 antibody raised against a keyhole limpet hemocyanin-conjugated peptide encompassing amino acids 360–382 of Cx43 followed by horseradish peroxidase-conjugated goat anti-rabbit IgG (1:1000; Amersham). Protein was visualized by autoradiography using ECL Western Detection Reagent (Amersham) and quantified by densitometry.
RNase Protection Assay
The RNase protection assays were performed as previously described by Fang et al. [24]. Samples (20 µg) of total RNA were hybridized to gel-purified antisense 32P-labeled rat OTR RNA probes in 80% formamide and 5-fold concentrated salts containing 200 mM PIPES, 2 M NaCl, and 5 mM EDTA for 18 h at 55°C. After incubation with 0.75 µg RNase A and 300 units RNase T1 (both from Boehringer Mannheim Canada, Laval, PQ, Canada) for 30 min at 30°C, protected fragments were analyzed on 6% denaturing polyacrylamide gels. A rat cyclophilin probe (Ambion, Austin, TX) was used in all samples as an internal control. The gel was exposed to x-ray film (Kodak X-Omat Blue) for the appropriate time, and resulting autoradiograms were quantified by densitometric analysis.
Statistical Analysis
All data were analyzed by two-way ANOVA followed by pairwise multiple comparison procedures (Student-Newman-Keuls method). The data for Figures 1B, 1D, 4B, and 5B were transformed to the natural logarithm (ln) to obtain a normal distribution. Statistical analysis was carried out using SigmaStat version 1.01 (Jandel Corp., San Rafael, CA) with the level of significance for comparison set at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The levels of OTR mRNA have also been shown to increase during late gestation in the rat myometrium [8]. In our vehicle-treated rats, a similar profile was observed, with significantly higher levels of OTR mRNA on Days 22 and 23L than on Days 20 and 21 (Fig. 2B). In PTHrP-treated rats, a similar delay in the gestational increase in OTR mRNA was observed as was shown for Cx43 mRNA and protein (Fig. 2A). In PTHrP-treated rats, OTR mRNA levels were 3-fold lower on Day 22 than those in vehicle-treated rats (Fig. 2B). Similar to the results for Cx43, on Day 23L there was no difference in the levels of OTR mRNA between the vehicle- and PTHrP-treated rats.
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Although treatment with PTHrP delayed the dramatic increase in CAP gene expression, it did not significantly affect the time of delivery, which was 4.67 ± 1.86 h (mean ± SEM) in the vehicle-treated group and 5.5 ± 1.04 h in the PTHrP-treated group after the lights were turned on in the animal colony at 0700 h on Day 23 of gestation. Because progesterone inhibits myometrial CAP gene expression, the maternal plasma progesterone concentrations in vehicle- and PTHrP-treated rats were measured. Treatment with PTHrP did not affect maternal plasma progesterone concentrations on any gestational day studied (Fig. 3). Progesterone concentrations in maternal plasma on Day 20 of gestation (93 ng/ml) were similar to those previously published [9]. As expected, progesterone concentrations in maternal plasma were significantly reduced on Days 22 and 23L compared with Day 20 in both the vehicle- and PTHrP-treated rats (Fig. 3).
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Results of previous studies have suggested that an increase in levels of the transcription factor c-fos is involved in the induction of Cx43 and OTR during labor [23]. To determine whether changes in c-fos levels could account for the reduced expression of Cx43 and OTR observed on Day 22 in PTHrP-treated rats, the mRNA levels of c-fos in vehicle- and PTHrP-treated pregnant rats were examined. Northern blot analysis indicated no difference in the c-fos mRNA levels between vehicle- and PTHrP-treated rats on any gestational day (Fig. 4). Previous gestational profile experiments have indicated that the increase in c-fos mRNA occurs only during labor. A significant increase in fra-2 mRNA was detected beginning on Day 21, compared with the level on Day 12, suggesting that fra-2 expression may be involved in inducing changes in CAP gene expression prior to the onset of labor [4]. Similar to c-fos mRNA, no significant difference was observed in the levels of fra-2 mRNA in PTHrP-treated animals on any gestational day (Fig. 5).
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Although the effects of PTHrP treatment on the contractile activity of the myometrium have been well characterized, expression of the PTH/PTHrP receptor has not been examined in the myometrium during late pregnancy. To determine whether the absence of an effect of PTHrP-treatment on CAP gene expression on Day 23L is due to withdrawal of the PTH/PTHrP receptor with the onset of labor, the mRNA levels of the PTH/PTHrP receptor in both vehicle- and PTHrP-treated rats were investigated. The results indicated no difference in the levels of PTH/PTHrP receptor mRNA in the myometrium of vehicle- and PTHrP-treated rats (Fig. 6), and there was no significant difference in PTH/PTHrP receptor mRNA levels among different late gestational days.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Treatment of pregnant rats with PTHrP delayed the dramatic increases in Cx43 and OTR concentrations during late gestation. The expression of both of these CAP genes enhances myometrial contractile activity. The expression of Cx43 in the myometrium allows for increased coupling of myometrial cells, thus allowing the myometrium to act as a functional syncitium and to contract spontaneously in a coordinated manner. The expression of OTR in the myometrium supports contractility by increasing the responsiveness of the myometrium to oxytocin. Therefore, in addition to the direct actions of PTHrP as a myometrial relaxant, this peptide may support myometrial quiescence through the inhibition of CAP gene expression.
Progesterone negatively regulates expression of both Cx43 and OTR throughout gestation; therefore, the actions of PTHrP could occur through altering the concentrations of this hormone. However, concentrations of maternal plasma progesterone were not altered by treatment with PTHrP, indicating that the actions of this peptide were through a separate mechanism. Thus, PTHrP may act in synergy with progesterone during late pregnancy to inhibit CAP gene expression. Because concentrations of progesterone begin to decrease on Day 20 of rat gestation, PTHrP may act to support myometrial quiescence and maintain low levels of CAP gene expression as the concentrations of progesterone decrease during late gestation [25]. Although our study did not provide evidence that the injected PTHrP acted directly on the myometrium, we did detect mRNA for the PTH/PTHrP receptor in the myometrium. In another study, PTHrP acted directly on excised uterine strips to inhibit contractions, indicating that PTHrP is able to act locally on uterine tissue [17]. Although members of the AP-1 family of transcription factors have been suggested to positively regulate expression of myometrial Cx43 during late pregnancy, the lack of any difference in c-fos and fra-2 mRNA levels with PTHrP treatment suggests that the actions of PTHrP were not through the inhibition AP-1 gene expression. In primary cultures of myometrial smooth muscle cells, PTHrP treatment caused an increase in intracellular levels of cAMP and the subsequent activation of PKA [26]. In the myometrium, PTHrP may act through the PKA pathway to directly or indirectly inhibit the expression of CAP genes.
Although PTHrP treatment significantly reduced the expression of Cx43 and OTR in the rat myometrium on Day 22, the levels of these CAP genes were not different on the day of labor and no effect on the timing of delivery was observed. In the human myometrium, PTHrP acts as a myometrial relaxant on tissue obtained before but not after the onset of labor [17], which suggests that with the onset of labor there is reversal of the mechanisms by which PTHrP reduces CAP gene expression. PTHrP is expressed in the rat myometrium throughout gestation; the levels peak on Day 22 and are not reduced with the onset of labor [13]. Thus, the ability of PTHrP to inhibit myometrial contractions and suppress CAP gene expression must be reversed or overridden to allow for the activation and stimulation of the myometrium.
The PTH/PTHrP receptor was detected in late pregnant rat myometrium by Northern blot analysis. The actions of PTHrP in the myometrium may therefore be mediated at least in part through this receptor. Treatment of human primary myometrial smooth muscle cells with PTHrP causes a dose-dependent increase in cAMP that is attenuated by PTH/PTHrP receptor antagonists, suggesting that the actions of PTHrP in myometrial cells are mediated by this receptor [26]. The absence of a change in the levels of mRNA encoding the PTH/PTHrP receptor with the onset of labor, however, suggests that withdrawal of PTHrP signaling is not through the inhibition of PTH/PTHrP receptor transcription. However, the levels of functional PTH/PTHrP receptor present in the cell membrane may be altered with the onset of labor. In a rat osteosarcoma cell line (ROS 17/2.8), receptor coupling to G-proteins was dependent on receptor density. At the highest receptor density, the PTH/PTHrP receptor was coupled to both Gs and Gq/11, at intermediate receptor densities only coupling to Gs was observed, and at the lowest receptor density no G-protein coupling was detected [20]. Therefore, in the myometrium a reduction in receptor density may lead to uncoupling of the PTH/PTHrP from Gs, preventing PTHrP from acting as a smooth muscle relaxant through the PKA pathway.
In addition to altering the levels of receptor expressed, the withdrawal of PTHrP signaling could occur through a reduction in the concentrations of G-proteins that couple to the PTH/PTHrP receptor or downstream signaling effectors. The increase in PKA activity as a result of PTHrP signaling in osteoblast cells is through receptor coupling to Gs and subsequent activation of adenylyl cyclase [20]. The reduction in the responsiveness of the rat myometrium to ß-adrenergic agonists at the end of gestation appears to occur through a dramatic reduction in expression and activity of adenylyl cyclase on the day before labor [27]. In the human myometrium, term pregnancy is associated with an increase in both Gs and Gs-coupled adenylyl cyclase activity [28]. This increase in G-protein expression could allow for the actions of PTHrP as both a myometrial relaxant and an inhibitor of CAP gene expression. With the onset of labor, there is a reduction in both Gs and Gs-coupled adenylyl cyclase activity, which could mediate the withdrawal of PTHrP signaling in the myometrium prior to the onset of labor in the absence of a change in receptor expression [28].
In addition to the actions of PTHrP as a myometrial relaxant during pregnancy, this peptide inhibits expression of the CAP genes Cx43 and OTR. PTHrP therefore supports myometrial quiescence through both immediate inhibition of myometrial contractility and through inhibiting expression of genes that support coordinated myometrial contractions. These actions of PTHrP in the myometrium may be important in allowing for maintained uterine quiescence as progesterone concentrations decrease during late gestation. The withdrawal of this PTHrP signaling occurs with the onset labor and may be an important step in the progression from the pregnant to the laboring myometrium.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Stephen J. Lye, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Ave., Suite 982, Toronto, ON, Canada M5G 1X5. FAX: 416 586 8857; lye{at}mshri.on.ca
Received: 11 February 2003.
First decision: 26 February 2003.
Accepted: 27 March 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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s in human myometrium in term and preterm labor: a mechanism for parturition. J Clin Endocrinol Metab 1994 79:1835-1839[Abstract]This article has been cited by other articles:
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  A. M. Flenniken, L. R. Osborne, N. Anderson, N. Ciliberti, C. Fleming, J. E. I. Gittens, X.-Q. Gong, L. B. Kelsey, C. Lounsbury, L. Moreno, et al. A Gja1 missense mutation in a mouse model of oculodentodigital dysplasia Development, October 1, 2005; 132(19): 4375 - 4386. [Abstract] [Full Text] [PDF]
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 V. Terzidou, S. R. Sooranna, L. U. Kim, S. Thornton, P. R. Bennett, and M. R. Johnson Mechanical Stretch Up-Regulates the Human Oxytocin Receptor in Primary Human Uterine Myocytes J. Clin. Endocrinol. Metab., January 1, 2005; 90(1): 237 - 246. [Abstract] [Full Text] [PDF]
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 J. E. I. Gittens, K. J. Barr, B. C. Vanderhyden, and G. M. Kidder Interplay between paracrine signaling and gap junctional communication in ovarian follicles J. Cell Sci., January 1, 2005; 118(1): 113 - 122. [Abstract] [Full Text] [PDF]
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