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Pregnancy |
Departments of Obstetrics & Gynecology3
Anatomy & Neuroscience,4 The University of Texas Medical Branch, Galveston, Texas 77555
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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neuropeptides, parturition, pregnancy, steroid hormones, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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synthesis also stimulate myometrial contractions and alter hormone synthesis at term [9]. In the pregnant rat, many of these changes appear to result from an increase in the estrogen:progesterone ratio at term. However, the significance of the factors that are involved in the maintenance of uterine quiescence during pregnancy before term is not well understood. These factors include nitric oxide (NO) and calcitonin gene-related peptide (CGRP). We and others have reported that NO is generated in the uterus and that both the production of NO and the relaxation sensitivity of uterus to NO are elevated during pregnancy and decreased at term [10–12]. The CGRP is a potent vasodilator [13]. Myometrial contractions are inhibited by CGRP in rats [14], humans [15], and mice [16], and these inhibitory effects of CGRP are substantially higher during pregnancy [14, 15]. At term during labor, the uterine relaxation sensitivity to CGRP is reduced, indicating a role for CGRP in maintaining uterine quiescence during pregnancy [14, 15]. In addition, serum CGRP levels were reported to increase during pregnancy and by sex steroid hormone treatment in female rats [17]. Changes in the myometrial relaxation sensitivity to CGRP appear to result from variations in its binding sites [14, 18]. Uterine CGRP-binding sites were elevated by progesterone and decreased by antiprogesterone [18], suggesting that the decline in CGRP-binding sites at term in rats may be related to the elevated estradiol:progesterone ratio. However, characterization of CGRP receptors and their changes in the uterus during pregnancy and labor are not well understood.
According to recent reports [19, 20], CGRP appears to mediate its effects by two receptors, CGRP-A and CGRP-B. The CGRP-B receptor [18, 21] was found to increase during pregnancy and to decrease at term labor [18] in rat uterus. The CGRP-A receptor consists of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying protein (RAMP) 1 [22]. On the other hand, CRLR, together with either RAMP2 or RAMP3, would function as a receptor for adrenomedullin (AM) [22]. In addition to CGRP, plasma levels of AM (a 52-amino acid, hypotensive peptide) were also reported to be elevated during pregnancy [23]. Furthermore, AM has been reported to inhibit galanin-induced contractions of rat uterus [24], suggesting its involvement in the relaxation of uterus during pregnancy. However, changes in CRLR, RAMP1, RAMP2, and RAMP3 in the rat uterus during pregnancy and parturition are not known. Therefore, the present study was designed to assess the changes in the mRNA expression of both CGRP-A- and AM-receptor components in the uterus during pregnancy and labor and by steroid hormone treatments in adult ovariectomized (ovx) rats.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult nonpregnant (body wt, 200–210 g) and time-mated pregnant Sprague-Dawley rats were purchased from Harlan Sprague Dawley (Houston, TX) and maintained on a 12L:12D photoperiod. Animals received an ad libitum supply of rat chow and water. Time-mated rats were killed on Day 18, during labor (one to three pups delivered), and on Day 2 postpartum. In addition, one group of nonpregnant rats at the diestrous stage was also killed. A group of pregnant rats on Day 18 were injected s.c. with a single dose of a progesterone antagonist, RU486 (10 mg/rat; Biomol, Plymouth Meeting, PA), and killed at 24 h after injection. Vehicle for steroid treatments (sesame oil; Sigma Chemical Co., St. Louis, MO) was injected (0.2 ml) into control animals.
In another set of experiments, virgin female adult rats underwent bilateral ovariectomy under general anesthesia using ketamine (45 mg/kg; Fort Dodge Laboratory, Fort Dodge, IA) and xylazine (5 mg/kg; Burns Veterinary Supply, New York, NY). Seven days later, the ovx rats received one of four different treatments for 3 days: 1) estradiol-17ß (2.5 µg/injection, twice daily s.c.); 2) progesterone (2 mg/injection, twice daily s.c.); 3) estradiol-17ß + progesterone (same dose and frequency as above); and 4) vehicle (sesame oil). All animals were killed in a CO2 inhalation chamber; the uteri were removed, cleaned, frozen immediately in liquid nitrogen, and stored at -70°C until used. All procedures were approved by the Animal Care and Use Committee of the University of Texas Medical Branch.
Isolation of Total RNA and Reverse Transcription
Total RNA was isolated from full-thickness uterine tissues using TRIzol (Life Technologies, Grand Island, NY). The quality and the quantity of the RNA were assessed at A 260/280, and all samples showed absorbency ratios ranging between 1.6 and 2.0. For reverse transcription (RT), 2 µg of total RNA were mixed with 3.0 nmol of random primer (Invitrogen, Carlsbad, CA), 200 µM dNTP solution (Sigma), and 10 U of AMV reverse transcriptase (Promega, Madison, WI) in the presence of 5 U of RNase inhibitor (Invitrogen) and placed in a thermal cycler for one cycle at 28°C for 15 min, 42°C for 30 min, 99°C for 5 min, and 4°C for 5 min.
Polymerase Chain Reaction
Polymerase chain reaction (PCR) of the cDNA was initiated by the published primers for CRLR and RAMP1 [22]. Primers for RAMP2 and RAMP3 were designed using their respective gene sequences from the GenBank database. Primer sequences are as follows: CRLR: forward, 5' TGCTCTGTGAAGGCATTTAC 3', and reverse, 5' CAGAATTGCTTGAACCTCTC 3'; RAMP1: forward, 5' GAGACGCTGTGGGTGACTG 3', and reverse, 5' TCGGCTACTCTGGACTCCTG 3'; RAMP2: forward, 5' GCTGTTACTGCTGCTGTTGC 3', and reverse, 5' GTCTGCCTCGTACTCCAAGC 3'; and RAMP3: forward, 5' CTTCTCCCTCTGTTGCTGCT 3', and reverse, 5' GTCCTGTCCACAGTGCAGTT 3'. Primers for 18S were obtained from Ambion, Inc. (Austin, TX). The PCRs were performed using 2.5 µl of cDNA. Complementary DNA was mixed with a PCR mixture containing 2.5 mM MgCl2, 1x PCR buffer, 200 µM dNTPs mix, and 300 nM forward and reverse primers for CRLR and RAMP1. For 18S, 2 µl of primer pair were used according to the supplier's specifications. The PCRs for CRLR, RAMP1, RAMP2, RAMP3, and 18S were carried out in a GeneAmp PCR system 9700 (Perkin-Elmer, Norwalk, CT) with the following conditions: An initial denaturation step at 95°C for 5 min was followed by 35 cycles of 30 sec at 95°C, 90 sec at 60°C, and 30 sec at 72°C. Reaction conditions for PCR of RAMP2 and RAMP3 were similar except that the annealing temperature was 63°C. All the reactions were terminated by a 7-min long elongation step at 72°C. Total cycle number was chosen for each gene from the linear portion of their respective curve.
Electrophoresis and Gel Imaging
The PCR products were visualized on 1.6% agarose gels containing 0.5 µg/ml of ethidium bromide and run for 1.5 h at 100 V in 0.5x Tris-borate-EDTA buffer. The DNA signals on the gel were imaged under ultraviolet light using a Polaroid camera (Photodyne, Inc., New Berlin, WI), and density-gradient measurements were performed using the FluorChem digital imaging system (Alpha Innotech Corporation, San Leandro, CA). The levels of expression of CRLR, RAMP1, RAMP2, and RAMP3 were calculated as a ratio to their respective 18S values. The identity of the amplified sequences was verified by sequencing the gel-extracted PCR product. All receptor components showed 100% homology with their respective published sequences (data not shown). Negative controls were run in PCR using total RNA in place of cDNA, and no signal was detectable when run on agarose gel (data not shown).
Statistical Analysis
Results are shown as the mean ± SEM of eight determinations. Statistical significance was determined by the Student t-test. A P value of less than 0.05 was considered to be statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression of CRLR, RAMP1, RAMP2, and RAMP3 mRNA in the rat uterus was determined by RT-PCR analysis, and the level of mRNA for these component proteins was determined relative to 18S mRNA in the uterus from each animal. Comparisons were made in the mRNA expression among groups of rats on Day 18 of gestation, during spontaneous labor at term, and on Day 2 postpartum and among nonpregnant rats. The CRLR mRNA in the rat uterus (Fig. 1) showed a significant increase (P < 0.001) during pregnancy compared to the nonpregnant levels. These levels declined significantly (P < 0.01) at term labor and on Day 2 postpartum compared to the pregnant levels. Expression of RAMP1 mRNA in the uterus was also significantly higher in pregnant (P < 0.01) compared to nonpregnant and in-labor rats. The RAMP2 mRNA levels in the uterus were significantly higher (P < 0.05) in pregnant compared to nonpregnant rats (Fig. 1). The levels of RAMP2 mRNA declined (P < 0.05) during labor compared to pregnant levels. The RAMP3 mRNA expression was significantly higher (P < 0.01) in pregnant compared to nonpregnant rat uterus (Fig. 1); however, the RAMP3 mRNA levels at labor were similar to the pregnant levels.
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Effect of RU486 on Uterine CRLR, RAMP1, RAMP2, and RAMP3 mRNA in Pregnant Rats
To assess the role of endogenous progesterone during pregnancy in the maintenance of mRNA for CRLR, RAMP1, RAMP2, and RAMP3, RU486 was injected s.c. on Day 18 of gestation. The levels of mRNA observed in the rat uterus were significantly lower for CRLR (P < 0.01) and for RAMP1, RAMP2, and RAMP3 (P < 0.05) at 24 h after RU486 treatment compared to the vehicle-treated controls (Fig. 2).
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Effect of Steroid Hormones on Concentrations of mRNA for CRLR, RAMP1, RAMP2, and RAMP3 in the Uterus of Nonpregnant Rats
To assess the pregnancy-independent effects of sex steroid hormones on uterine mRNA levels for CRLR, RAMP1, RAMP2, and RAMP3, adult ovx rats were treated with progesterone, estrogen, or estrogen in combination with progesterone. Progesterone treatment in adult ovx rats caused significant elevation in mRNA expression for CRLR (P < 0.01) and for RAMP1 and RAMP2 (P < 0.01), but not for RAMP3 (Fig. 3). Estradiol-17ß treatment, on the other hand, caused inhibition in the expression of mRNA for CRLR (P < 0.01) and for RAMP1, RAMP2, and RAMP3 (P < 0.05). The estradiol-17ß-induced decline in mRNA expression of CRLR, RAMP1, RAMP2, and RAMP3 in the ovx rats was also significant (P < 0.05) compared to the expression in progesterone- or in progesterone- and estradiol-17ß-treated rats.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression of the CGRP-A-receptor components, CRLR and RAMP1, in rat uterus was increased during pregnancy and decreased at labor in the present study. These increases paralleled the reported changes in CGRP-induced inhibitor effects on uterine contractility [14]. Binding of [125I]CGRP also increased during pregnancy and decreased at term labor, suggesting that the functional receptors for CGRP are increased during pregnancy and decreased at term labor [18]. Decline in the CGRP receptor-related inhibitory effects of CGRP on contractility [14] coincides with the upregulation of stimulatory factors of uterine contraction [6, 8, 9] at term. Expression of CRLR and RAMP1 mRNA was increased during pregnancy and decreased during spontaneous labor at term as well as during RU486-induced preterm labor, suggesting that both the components of CGRP-A receptor, CRLR and RAMP1, are regulated during pregnancy and labor. Dynamic changes in the estrogen:progesterone ratio occur at term in pregnant rats. Increases in estrogen with decreases in progesterone may trigger the decline in CGRP-A receptors, thus facilitating labor and delivery of the fetus.
Progesterone treatment caused significant increases in CRLR and RAMP1 expression, whereas estrogen treatment decreased CRLR and RAMP1 mRNA expression, in adult ovx rats in the present studies. Binding of [125I]CGRP also increased in ovx rats treated with progesterone [18], suggesting that progesterone may be responsible for the upregulation of CGRP-receptor expression during pregnancy. Decreases in CRLR and RAMP1 mRNA levels with estrogen in the present study further supports the hypothesis that elevated estrogen with a fall in progesterone levels at term may induce substantial decreases in CGRP-A-receptor concentration in the uterus of pregnant rats. Therefore, CGRP-A receptors appear to play an important role in maintaining quiescence of uterus during pregnancy, when their expression is elevated, and by decreasing CGRP-induced inhibition of uterine contractility at term, when their expression is decreased.
Previous studies using monoclonal antibody to affinity-purified CGRP-receptor protein (CGRP-B) from porcine cerebellum [25], which is unrelated to CRLR [19, 20, 26], showed that CGRP-B receptors in the rat uterus increase during pregnancy and decrease at term [18]. Progesterone treatment of adult ovx rats caused an increase in these receptors in the uterus, whereas estrogen had no significant effects. These changes in CGRP-B receptors in the uterus of pregnant and progesterone-treated adult ovx rats are similar to the changes in CGRP-A-receptor mRNA levels observed in the present investigation. Increase in CGRP-A (present study) and -B [18] receptors observed in pregnant and in progesterone-treated adult ovx rats suggests that progesterone upregulates both these receptors during pregnancy and, therefore, that progesterone could be responsible for maintenance of CGRP-related uterine quiescence during pregnancy. Additionally, an increase in estrogen at term could induce a fall in CGRP-A receptors in the uterus and, thus, attenuate CGRP-related uterine relaxation and facilitate labor and delivery.
In association with either RAMP2 or RAMP3, CRLR forms an AM receptor [22]. In the present study, both RAMP2 and RAMP3 mRNA levels were increased during pregnancy. Whereas RAMP2 expression decreased at labor, RAMP3 expression was not different from the pregnant level. Treatment with RU486 in pregnant rats decreased the expression of both RAMP2 and RAMP3 mRNA. Although progesterone treatment was without significant effect on RAMP3, it increased RAMP2 expression in ovx rats. Because CRLR can form AM receptors with either RAMP2 or RAMP3, we suggest that AM could also play a role in maintaining uterine relaxation during pregnancy. In support of a role for AM in uterine relaxation, the plasma levels of AM were also reported to increase during pregnancy [23]. Specific and pharmacologically distinct binding sites were reported for both AM and CGRP in rat uterus [24]. The density of these AM-binding sites in the uterus were found to be approximately 10-fold higher in the pregnant compared with the nonpregnant rats, indicating pregnancy-related regulation of AM receptor. Galanin-induced uterine contractions were inhibited by both CGRP and AM [24], suggesting their involvement in uterine relaxation during pregnancy. However, in the same study, effects of both CGRP and AM are reported to be abolished by pretreatment of rat uterine tissue with CGRP8–37, suggesting that the contractile effects of both peptides are mediated by a related receptor or receptor component(s). Because CRLR is the common component of both CGRP-A and AM receptors, CRLR may play a significant role in uterine relaxation during pregnancy. The RAMP1 and RAMP2 mRNA levels in the uterus were also elevated both during pregnancy and with progesterone treatment and were decreased with term and RU486-induced preterm labor, further supporting roles for both CGRP and AM in the regulation of uterine contractility. The present studies further suggest that progesterone-related increases in both CGRP-A and AM receptors in the rat uterus may be involved in maintaining uterine quiescence during pregnancy. Decreases in progesterone and increases in estrogen levels at term could downregulate these receptors in the uterus and, thus, facilitate initiation of labor and delivery.
Although the present study clearly demonstrates the pregnancy- and parturition-related changes in mRNA for CRLR and RAMPs in rat uterus, it is unclear if these changes in mRNA reflect in the protein levels and in their location in the uterus. Our previous report indicated that myometrium appeared to be the primary site for abundant CGRP-binding sites [18]. However, further studies are required to fully assess the changes in proteins for various receptor components and their location in the uterus.
In summary, both CGRP-A- and AM-receptor components in the rat uterus are elevated during pregnancy and are decreased at term and RU486-induced preterm labor. Progesterone increased, whereas estrogen inhibited, the levels of these receptors, indicating regulation by steroid hormones. Therefore, changes in both CGRP-A- and AM-receptor levels in the uterus may play a role in maintaining uterine quiescence during pregnancy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Chandrasekhar Yallampalli, The University of Texas Medical Branch, Department of Obstetrics & Gynecology, 301 University Boulevard, Galveston, Texas 77555-1062. FAX: 409 747 0475; chyallam{at}utmb.edu
Received: 17 January 2003.
First decision: 10 February 2003.
Accepted: 23 May 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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