Departments of 1 Medicine and 2 Obstetrics and Gynecology, Christchurch School of Medicine, Christchurch 8001, New Zealand; and 3 Baker Medical Research Institute, Prahran, Victoria 8008, Australia
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ABSTRACT |
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Levels of expression of adrenomedullin (AM) in the uterus have been reported to vary with the reproductive cycle. This study examines the relationships among uterine AM mRNA, the stage of the estrous cycle, and circulating estradiol and progesterone in cycling rats and in ovariectomized (OVX) rats without or with estrogen replacement (ER). Strong AM mRNA, AM immunoreactivity, and pro-AM NH2-terminal 20 peptide (PAMP) immunoreactivity were observed in endometrial stroma by use of in situ hybridization and immunocytochemistry. Endometrial expression was particularly intense at proestrus and estrus, with weaker expression in the myometrium. By RNase protection assay, significant differences in AM mRNA between the stages of the estrous cycle could not be established. However, levels of AM mRNA were positively correlated with plasma estradiol in cycling rats (r = 0.56, P < 0.005) and in OVX and ER rats (r = 0.92, P < 0.001) and were not correlated with plasma progesterone. Levels of AM mRNA were significantly reduced after OVX compared with cycling rats, and ER restored AM mRNA to levels equivalent to those seen at the peak of the cycle (proestrus). In conclusion, although AM expression in the uterus varies throughout the estrous cycle, it is more closely correlated with circulating estradiol levels than with the stage of the cycle itself.
adrenomedullin; proadrenomedullin NH2-terminal 20 peptide; estradiol; uterus; estrous cycle
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INTRODUCTION |
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ADRENOMEDULLIN (AM) was originally identified in pheochromocytoma tissue by its ability to elevate cAMP in rat platelets (10). The mature AM peptide consists of 52 amino acids in humans (19) and pigs (18) and 50 amino acids in the rat (31) and mouse (30). The sequence of AM bears some homology with calcitonin gene-related peptide (CGRP) and amylin. When AM is injected into animals, its biological actions include vasodilation and a fall in blood pressure (17, 27, 28), in part via generation of nitric oxide (24). In cultured vascular smooth muscle cells, AM is antiproliferative and stimulates cAMP (14). In addition to AM, the proadrenomedullin gene codes for a second biologically active peptide (16), named proadrenomedullin NH2-terminal 20 peptide (PAMP), which also reduces arterial pressure in the rat (16). AM is thought to act via specific G protein-coupled receptors with seven transmembrane domains, and there is some cross talk between AM and CGRP receptors (10).
AM is highly expressed in the human (21) and rat (4) female reproductive tracts. In the uterus, both AM immunoreactivity (AM-IR) (21) and AM mRNA (4) have been detected in the endometrium and, to a lesser extent, in the myometrium. Endometrial AM-IR reportedly varies during the human reproductive cycle (20, 21), but one study observed stronger AM-IR staining during the proliferative phase (21), whereas the other reported AM-IR to be stronger during the secretory phase (20). Although AM expression has been shown to be induced in human endometrial stromal cells by the nonsteroidal antiestrogen tamoxifen, estradiol itself did not affect AM expression in these cells (34). To clarify the role of the reproductive cycle and the relationship between reproductive hormones and regulation of AM expression in the uterus, we have assessed levels of AM mRNA throughout the rat estrous cycle. The pattern of AM expression in the uterus was visualized using in situ hybridization and immunocytochemistry, and levels of AM mRNA were quantified using the RNase protection assay. To investigate the role of reproductive hormones in uterine AM expression, the relationships between circulating estradiol and progesterone and AM mRNA were analyzed among cycling rats and in ovariectomized rats with and without estrogen replacement.
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MATERIALS AND METHODS |
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Collection of rat tissues.
Adult female Sprague-Dawley rats (170-250 g) were housed six
per cage under 12:12-h light-dark cycles. The stage of the estrous cycle was determined by cytology of daily vaginal smears examined with
Pasini staining. After rats were followed through at least three
cycles, they were killed at a predetermined stage of the cycle either
for in situ hybridization or for RNase protection assay (RPA). To
compare the effect of ovariectomy (OVX) with OVX plus estradiol
replacement (ER), an additional group of rats underwent OVX via
bilateral flank incisions under anesthesia induced by Nembutal (40 mg/kg body wt ip). For the ER group, a slow-release pellet containing
-estradiol (0.5 mg/pellet; Innovative Research of America, Sarasota,
FL) was implanted subcutaneously at the nape of the neck at the time of
surgery. Placebo pellets were implanted in the OVX group.
In situ hybridization and immunohistochemistry.
Riboprobes for in situ hybridization were generated by in vitro
transcription from murine AM DNA templates, as previously described
(4). Briefly, a probe of 555 bp, referred to as the pro-AM
probe, was generated by RT-PCR from mouse kidney RNA using the primers
AM-B and AM-3' given below. This pro-AM probe spans the regions coding
for both AM and PAMP (nucleotides 53-607 of the mouse cDNA
sequence in the DGBJ/EMBL/Genebank data libraries, accession number
U77630). The relative identity between the cDNA sequences of mouse and
rat in the region of the pro-AM probe is 90%. To generate the cDNA
probe, total mouse kidney RNA was prepared by TRIzol (GIBCO-BRL, Grand
Island, NY) extraction, reverse transcription was performed with
Superscript II (GIBCO-BRL) with an oligo-dt primer, and then PCR was
performed with the following primers
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RPA. Total RNA was extracted from the uterine samples (~10 mg tissue) using TRIzol, according to the manufacturer's instructions. A 613-bp fragment of rat AM complementary (c)DNA was synthesized from rat adrenal gland, as previously described (3). The cloned rat AM cDNA fragment was used as a template to generate 32P-labeled complementary RNA probes for use in solution hybridization/ribonuclease protection analysis, as described previously (3). A rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe was used as a control probe. Rat uterus total RNA (5 µg) was hybridized with both AM and GAPDH probes, and the protected RNA hybrids were analyzed on nondenaturing polyacrylamide gels and quantitated on a Fuji BAS-1000 (Tokyo, Japan) phosphorimaging system. Concentrations of AM mRNA were corrected for relative GAPDH concentrations to allow for minor variations in RNA loading and variability of the cellular transcriptional activity in the samples. This was performed by assigning the first GAPDH sample a value of 1.0 and multiplying each AM mRNA value by its relative GAPDH value.
Plasma estradiol and progesterone assays.
Concentrations of plasma estradiol were measured using a
radioimmunoassay kit, the 17-estradiol2 Sensitive Kit
(Sorin, Saluggia, Italy). The assay has a detection limit of 5 ± 1, an interassay coefficient of variation (CV) of 5.2% at 100 pmol,
and an intra-assay CV of 3.8%. Concentrations of progesterone were
measured in plasma by ELISA with a monoclonal antibody, by use of a
previously published method (7).
Statistics. Relative levels of AM mRNA corrected for GAPDH were compared across the different treatment groups by one-way ANOVA. Post hoc comparisons between treatment groups were then conducted using Fisher's least significant difference test. Correlations between the concentrations of AM expression, plasma estradiol, and plasma progesterone were calculated using two-tailed Pearson's correlation coefficients, with log plasma estradiol values to normalize the data. Statistical significance was taken at the P < 0.05 level.
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RESULTS |
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Uterine tissue collected from each stage of the rat estrous cycle
was examined by in situ hybridization, and representative sections are
shown in Fig. 1. Strong AM mRNA was
observed in the endometrial stroma, particularly at proestrus and
estrus, with the most intense AM mRNA surrounding the glands. Weaker AM
expression was observed in the outermost layer of the myometrium,
which, in contrast to the endometrium, did not vary appreciably between different stages of the estrous cycle. Immunostaining with an AM
antibody in the uterus throughout the cycle demonstrated an intensity
similar to that of AM mRNA, being strongest at proestrus and estrus and
weakest at diestrus (representative sections are shown in Fig.
2). The immunostaining obtained using an
antibody to PAMP (PAMP-IR, Fig. 2), displayed a very similar pattern to that with the AM antibody, with the strongest staining in endometrial stromal cells, especially those surrounding the glands.
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The concentrations of AM mRNA and GAPDH were quantified by RPA, as
illustrated in Fig. 3A. To
allow for interanimal variation and minor disparities in RNA loading,
the AM mRNA data were corrected for relative GAPDH expression. The
concentration of GAPDH expression was not significantly associated with
the stage of the estrous cycle, plasma estradiol, or plasma
progesterone. However, there was an indication that GAPDH was increased
in ER rats compared with OVX rats, suggesting that the transcriptional
activity of the tissue can vary in such nonphysiological states.
Correcting the AM mRNA for relative GAPDH levels controlled for any
variation in transcriptional activity between treatment groups.
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Data obtained by RPA indicated that there was a wide scatter of AM mRNA values through the cycle (Fig. 3A), and significant differences in mean AM expression between different stages of the estrous cycle could not be established [F ratio = 0.16, degrees of freedom (df) 2,16, P = 0.85]. Levels of AM mRNA in the OVX group were significantly reduced compared with the peak of the cycle at proestrus (P = 0.025, Fig. 3B). ER in OVX rats restored AM mRNA to levels equivalent to those seen at the peak of the cycle (Fig. 3A, no significant difference between proestrus and ER groups). Levels of AM mRNA in the ER rats were significantly greater than in the OVX rats (P < 0.0001).
Levels of plasma estradiol throughout the estrous cycle are summarized
in Table 1. Plasma estradiol in the ER
group was significantly greater than in all other treatment groups, but
there were no other significant differences among the groups. Levels of
AM mRNA were positively correlated with plasma estradiol within the
group of cycling rats (r = 0.56, n = 19, P < 0.005) and within the group of OVX and ER-treated
rats (r = 0.92, n = 15, P < 0.001). Levels of uterine AM mRNA were also positively correlated with
plasma estradiol (Fig. 3B) when data from all rats were
combined (r = 0.46, P < 0.005).
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Levels of plasma progesterone were significantly different across the
estrous cycle (Table 1), with highest levels at estrus and lowest at
proestrus (significance between these groups, P = 0.03). Progesterone levels in the OVX and ER groups were significantly lower than both the diestrus and estrus levels (P < 0.001). Concentrations of plasma progesterone in cycling rats were
negatively correlated with the raw data of AM mRNA in uterus (r
= 0.529, P < 0.02). However, when these AM mRNA
levels were corrected for GAPDH expression, they were not significantly
correlated with plasma progesterone.
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DISCUSSION |
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This study is the first quantitative analysis of AM mRNA expression in uterus throughout the reproductive cycle. In the rat, a direct relationship between levels of AM mRNA and the stage of the cycle could not be established, although there was a general trend for endometrial AM expression to peak at proestrus and estrus. However, levels of uterine AM mRNA were significantly correlated with circulating estradiol concentrations. We conclude that, although AM expression in the uterus varies throughout the estrous cycle, levels of AM mRNA are more closely correlated with circulating estradiol concentrations than with the stage of the cycle itself.
Previous studies have found AM to be highly expressed in uterine tissue of the human (21) and rat (4), and relative staining of AM-IR has been observed to vary during the reproductive cycle in humans (20, 21). In the present study, we failed to confirm a direct relationship between the stage of the estrous cycle and AM mRNA expression. However, using three different methods for monitoring AM expression, we observed that those individuals with the highest levels of uterine AM mRNA, AM-IR, and PAMP-IR were in proestrus or estrus, and that these high levels of AM expression were associated with high levels of circulating estradiol. Moreover, whereas OVX almost totally abrogated uterine AM expression, estradiol replacement in OVX rats restored AM mRNA to levels equivalent to those seen in intact rats at the peak of the cycle, proestrus. This suggests that estradiol is capable of inducing uterine AM expression, but whether this is a direct or indirect effect is yet to be determined.
Because the secretion of plasma estradiol is related to the stage of the estrous cycle, the observation that AM mRNA was correlated with plasma estradiol concentrations but not the stage of the estrous cycle appears contradictory. However, in the rat there is considerable variability in the secretion of estradiol during the estrus cycle (32), with a major peak of estradiol in late proestrus but also occasional spikes of secretion throughout the cycle. The variability of this pattern of secretion in the rat differs from that in the human menstrual cycle, in which a sharp midcycle peak is seen immediately before ovulation and a secondary peak is seen around the middle of the luteal phase (9). The present study suggests that there are surges of AM mRNA associated with spikes of estradiol throughout the rat estrous cycle, and this may account for our inability to establish significant differences in AM mRNA among different stages.
A previous study using differential PCR display demonstrated that, in cultured human endometrial stromal cells, estrogen did not induce the expression of AM mRNA, whereas the nonsteroidal antiestrogen tamoxifen induced AM mRNA in these cells (34). These data, in combination with our findings, suggest that estradiol may have an indirect effect on uterine AM mRNA, interacting with other factors to influence AM expression. In contrast to estradiol, there was a weak inverse relationship between circulating progesterone concentrations and uterine AM expression, apparent only in the raw data and not when AM mRNA was corrected for GAPDH. This weak relationship probably resulted from the fact that rising levels of circulating progesterone levels coincide with falling levels of circulating estradiol during the estrous cycle (32), rather than being a direct effect of progesterone on uterine AM mRNA. Therefore, it is unlikely that progesterone plays a major role in regulating uterine AM mRNA during the estrous cycle. Because we found that estradiol replacement in OVX rats was associated with raised AM mRNA in the absence of ovarian function, factors involved in the interaction between estradiol and AM expression are unlikely to be ovarian in origin. It is likely that local factors within the uterus either are produced in response to estradiol or synergize with estradiol to enhance AM expression.
No previous studies have examined the regulation of AM production by
the uterus. However, in other tissues such as vascular smooth muscle
cells, fibroblasts, and cardiac myocytes, AM production has been shown
to be increased by a range of cytokines, growth factors, and hormones
(10). These include tumor necrosis factor- and -
,
interleukin-1
and -
, dexamethasone, cortisol, aldosterone, retinoic acid, and thyroid hormones. Other studies have demonstrated that oxidative stress in vascular smooth muscle cells (1)
and hypoxia in human umbilical vein endothelial cells (29)
can increase AM production. One cytokine that displays coordinate
expression with AM in both the mouse placenta and rodent embryos is
transforming growth factor-
1 (TGF-
1) (26).
Endometrial stromal cells also express TGF-
1 (2), and
estradiol-17 increases levels of TGF-
1 mRNA in these cells in
culture. Although those data suggest that TGF-
1 may be a candidate
for mediating the effects of estradiol on AM production, TGF-
1 has
been reported to suppress AM secretion in rat endothelial cells
(11). The effects of TGF-
1 on AM expression in uterine
endometrium have yet to be investigated.
It has been proposed that the role of AM in the uterus is to inhibit muscular tone of the myometrium. AM has been reported to attenuate the contraction of isolated uterine smooth muscle induced by galanin (33), in keeping with the effects of AM to relax smooth muscle in the respiratory tract (13) and vascular tissues (28). Levels of AM in uterine tissue (33) and in plasma (6) are raised during pregnancy, consistent with the hypothesis that AM may have a role to suppress uterine contractility. Alternatively, AM may have a role in regulation of the blood supply or proliferation of vascular tissue during the reproductive cycle. In sheep uterus, AM has been shown to be a potent vasodilator, eliciting increased uterine blood flow (8). This study found that AM was 16 times more potent than prostaglandin I2 (PGI2) as a vasodilator in the uterine circulation. Furthermore, AM has been shown to be angiogenic in the chick chorioallantoic membrane assay (34). Taken together, those findings suggest that AM secreted by the endometrium may participate in regulating angiogenesis in the proliferating endometrium and help control uterine blood supply during the estrus cycle. In light of the multifunctional nature of AM in the many tissues where it is located, it is possible that AM has dual roles in the uterus, regulating uterine tone within the myometrium and regulating angiogenesis and blood supply within the endometrium.
In addition to the role of AM in the nonpregnant uterus, pregnancy and sepsis are the two physiological conditions in which AM is consistently increased (10). Because levels of AM mRNA are significantly raised in pregnancy (33), it has been suggested that the uterus itself may be the source of circulating AM. Moreover, in a rat model of preeclampsia induced by NG-nitro-L-arginine methyl ester, infusion of AM reversed hypertension and reduced pup mortality when given to rats in late gestation (22). In mice with the AM gene deleted, embryos die in midgestation, exhibiting extreme hydrops fetalis with fluid-filled thoracic cavities (5). It would appear that AM plays a vital role in fluid balance during both pregnancy and embryogenesis.
The distributions of staining observed with the AM and PAMP antibodies were very similar, suggesting that these two peptides are cotranslated in uterine tissue. The immunostaining of both AM and PAMP was observed in the striatum spongiosum and striatum compactum layers of the endometrium, and it was more intense around the endometrial glands. This distribution may indicate an angiogenic role for these peptides in the formation of the rich capillary plexus around the glands, a process which is highly responsive to the hormonal changes of the estrous cycle. Alternatively, it is possible that a factor released from the glands induces AM production, and hence a gradient of intensity of peptide immunostaining can be observed around the glands.
It is likely that the AM produced by the uterus has a local site of action and is not secreted into plasma, because levels in human plasma do not correlate with either the menstrual cycle or with circulating estradiol (23). Consistent with this proposal, specific binding sites for AM have been identified in rat uterus (33), and the density of these sites is 10 times greater in pregnancy. A single AM receptor (15) has been cloned from rat adrenal and identified as a member of the seven-transmembrane, G protein-linked receptor superfamily. Messenger RNA for the AM receptor has been detected in rat uterus (33). However, in many tissues, the binding of AM is displaced by CGRP, indicating that AM cross-reacts with CGRP receptors. Recently, a family of receptor activity-modifying proteins (RAMPs) have been cloned that associate with the calcitonin receptor-like receptor (CRLR), presenting it at the cell surface as either an AM or a CGRP receptor (25). When RAMP2 transported the CRLR to the cell surface, the complex was shown to act as a specific AM receptor (25). Specific binding sites for PAMP, a second translated product of the AM gene, have also been demonstrated in aorta, adrenal glands, and at lower levels in other tissues (12), but binding of PAMP to uterine tissue was not examined. Until the entire family of AM receptors has been characterized, only tentative conclusions can be made about the tissue-specific actions of AM.
Overall, although a direct relationship between levels of AM mRNA and the stage of the cycle could not be established in this study, levels of uterine AM mRNA were significantly correlated with circulating estradiol concentrations. Thus AM expression in the uterus appears to be more closely correlated with circulating estradiol levels than with the stage of the cycle itself. The regulation of AM in uterus during the reproductive cycle and pregnancy is consistent with a reported effect of AM to inhibit smooth muscle contractility, adding increasing evidence to the proposal that AM has a role in reproductive function. These findings suggest that, in addition to being a cardiovascular hypotensive hormone, AM may have multiple endocrine roles.
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ACKNOWLEDGEMENTS |
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We thank Tom Pilling and Rachel Brennen (Christchurch School of Medicine) for care of the animals.
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FOOTNOTES |
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This research was supported by a Programme Grant from the Health Research Council of New Zealand and grants from the Canterbury Medical Research Foundation and Lotteries Grants Board.
Address for reprint requests and other correspondence: V. Cameron, Dept. of Medicine, Christchurch School of Medicine, PO Box 4345, Christchurch 8001, New Zealand (E-mail: vicky.cameron{at}chmeds.ac.nz).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 10 April 2001; accepted in final form 28 August 2001.
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