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Original Studies |
Sir Quinton Hazell Molecular Medicine Research Center, Department of Biological Sciences, University of Warwick, Coventry, United Kingdom CV4 7AL
Address all correspondence and requests for reprints to: Prof. E. W. Hillhouse, Sir Quinton Hazell Molecular Medicine Research Center, Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry, United Kingdom CV4 7AL.
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Abstract |
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, 1ß, 2, and the variant C, of the CRH receptor.
Interestingly, the CRH receptor subtypes in myometrium exhibit
differential expression patterns; in human pregnant myometrium at term
all four receptor-subtypes were expressed, whereas only the 1- and
1ß-receptor subtypes were found in the nonpregnant myometrium. This
would suggest that CRH, acting via different receptor subtypes, is able
to exert different actions on the myometrium in the pregnant state
compared to the nonpregnant state. Furthermore, in the pregnant human
uterus, CRH receptors were localized in both smooth muscle and
fibroblasts. These findings suggest that CRH receptor expression plays
an important modulatory role in myometrial and possibly in cervical
function. |
Introduction |
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The mRNAs encoding distinct CRH receptors (R1 and R2) have been cloned
(6, 7) and have been shown to arise from separate genes. mRNAs from
each of these genes exist as alternatively spliced forms, each giving
rise to at least two protein variants, and ß. The proteins
encoded by the R1 and R2 genes share 70% identity at the amino acid
level, although the biochemical consequences of these differences both
between receptor types and between variants of each type is not known.
In addition, a subtype, C, of the CRH-R1 receptor has been identified
by complementary DNA (cDNA) cloning from the human hippocampus (8). The
CRH-R2 receptor has a higher affinity for sauvagine, whereas the CRH-R1
receptor responds equally to both CRH and sauvagine in terms of its
ability to stimulate cAMP production (9). Recently, we have reported
that the human placenta and fetal membranes at term express the 1,
1ß, and 1C receptor subtypes.
The purpose of this study was to localize the CRH receptor mRNA in myometrial tissue obtained from both pregnant (term) and nonpregnant women.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Pregnant myometrial biopsies (n = 10) were obtained from women undergoing elective caesarean section at term before the onset of labor for nonmaternal problems. The biopsy site was standardized to the upper margin of the lower segment of the uterus in the midline. This provides the closest approximation to the upper segment of the uterus. Nonpregnant myometrial tissues (n = 8) were obtained from premenopausal controls undergoing hysterectomy for nonmalignant conditions. The nonpregnant myometrial biopsies were obtained from the same location as the caesarean section myometrial biopsies to avoid possible differences in receptor expression patterns. The relative contents of myometrial and fibrous tissues in these biopsies were identified by immunostaining using specific smooth muscle cell and fibroblast markers (actin and vimentin, respectively). The biopsies were immediately snap-frozen in liquid nitrogen and subsequently stored at -70 C until use. Ethical approval was obtained from the local ethical committee, and informed consent to the study was obtained from all patients.
Chemicals
Mouse monoclonal vimentin antibody and antimouse
IgG-tetramethylrhodamine isothiocyanate (TRITC) conjugated were
obtained from Sigma Chemical Co. (Poole, UK). Mouse monoclonal muscle
actin antibody was obtained from Dako (High Wycombe, UK). PCR and
cloning reagents were purchased from Life Technologies (Renfrewshire,
UK), and the DNA sequencing kit and [-35S]ATP were
obtained from Amersham International (Aylesbury, UK). The DNA 3'-end
labeling kit was purchased from Boehringer Mannheim (Bell Lane, UK).
Synthetic oligonucleotide probes and enzymes were purchased from Life
Technologies (Paisley, UK). Fluorescein isothiocyanate
(FITC)-conjugated antigoat IgG and the specific CRH receptor antibody
that recognizes both human CRH-R1 and CRH-R2 receptors were obtained
from Santa Cruz Biotechnology (Santa Cruz, CA). This is a goat
polyclonal antibody raised against a peptide corresponding to amino
acids 425–444 mapping at the C-terminus of the human CRH-R1 precursor.
All other chemicals were purchased from BDH (Poole, Dorset, UK).
Fluorescent in situ hybridization (FISH)
Paraffin-embedded blocks of human myometrium previously fixed in 4% (wt/vol) paraformaldehyde in 10 mmoL/L phosphate-buffered saline (PBS) were sectioned (7 mm) and mounted onto gelatin-coated slides. At the time of hybridization, sections were dewaxed using xylene, dehydrated by successive washes through ethanol, and air-dried. Synthetic oligonucleotide probes (Life Technologies) with fluorescein conjugated at their 5'-end were used in this study.
After prehybridization, sections were covered with parafilm and incubated at 37 C for 5 h in a humidified chamber. Then hybridization solution (100 mL) containing 25% formamide, 4 x SSC (standard saline citrate), 5% dextran sulfate, 0.2% dried milk powder, and 1 ng/mL probe was added, and the section was sealed with Sureseal (Hybaid, Teddington, UK) and allowed to hybridize at 37 C for 20 h. After hybridization, sections were washed twice with 2 x SSC at 45 C and once with 0.1 x SSC at 45 C. The sections were then rinsed in PBS, air-dried, and visualized under a fluorescent microscope. Samples of pregnant and nonpregnant human myometria were probed with both sense and antisense oligonucleotide probes of CRH receptors to ensure specificity.
RT-PCR and cloning
Polyadenylated RNA was isolated from pregnant and nonpregnant
myometrium tissues and reverse transcribed to synthesize cDNA; this was
used as a template for the first round of PCR. The products of the
first PCR reaction served as a template for the second round of
amplification reaction. Specific primers for the different CRH receptor
subtypes, as listed in Table 1
, were used in the second round of PCR.
The products of the second PCR reaction were subcloned and sequenced
(see below). As a negative control for all reactions, distilled water
was used in place of the cDNA.
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The PCR products were purified from a 1.0% agarose gel and ligated using a T4 DNA ligase kit (Life Technologies) into plasmid pBluescript II SK+/-. Positive isolated clones were sequenced, using internal primers for the whole gene, in an automated DNA sequencer, and the sequence data were analyzed using Blast Nucleic Acid Database Searches from the National Center for Biotechnology Information (Washington, DC).
Immunofluorescence
Paraffin-embedded sections of myometrial pregnant and nonpregnant tissues were dewaxed and dehydrated using the same procedure as that used for FISH. Specimens were incubated with PBS containing 3% BSA for 1 h (to block nonspecific binding) before addition of the primary goat polyclonal CRH-R1 antibody, which was used at a 1:100 dilution. All dilutions were made in 3% BSA in PBS. Specimens were incubated with the primary antibody (goat anti-CRH-R1/2) for 60 min and then washed three times with PBS before incubation with the second primary antibody (mouse antiactin or vimentin) for 30 min. After three washes with PBS, the sections were incubated with the first antigoat IgG-FITC secondary antibody for 2 h at room temperature. The sections were then incubated with the second antimouse IgG-FITC secondary antibody for 30 min and mounted on slides using 90% glycerol-PBS.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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To detect the CRH receptor subtypes, different specific primers
were used (Table 1
) in a nested PCR. When
primers 2S and 2A were used for the nested PCR, two DNA fragments with
sizes of 1.07 and 1.18 kb were amplified from the pregnant myometrium,
whereas only one fragment of 1.18 kb was amplified from the nonpregnant
myometrium (Fig. 1). These two DNA
fragments were subcloned into pBluescript II+/- and
sequenced. The nucleic acid sequence of the fragments revealed that in
both tissues, the 1.18-kb fragment corresponded to the human CRH-R1
receptor, whereas the 1.07-kb fragment amplified from pregnant
myometrium was from the CRH-R1C receptor. In addition, one DNA fragment
of 0.8 kb was amplified from the pregnant myometrium only. Nucleotide
sequencing revealed that this had been generated by nonspecific
annealing of the PCR primers, indicating the importance of nucleotide
sequencing of the PCR products to confirm the identity of the amplified
DNA.
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).
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could be detected in human myometrium,
specific primers were designed (Table 1) and used for a nested PCR. A
single strong band of 1.2 kb was amplified from the pregnant
myometrium, but not from the nonpregnant myometrium (Fig. 3). This was confirmed as being derived
from CRH-R2 by nucleotide sequencing.
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FISH and immunofluorescence
After RT-PCR and identification of CRH receptor mRNA transcripts,
we used FISH to localize the cellular distribution of the CRH-R
subtypes by using oligonucleotide probes specific for each type of
receptor mRNA. This identified CRH-R1 (Fig. 4, B–D) and CRH-R1ß (data not shown)
receptor mRNA in both pregnant and nonpregnant myometria, whereas
CRH-R1C (data not shown) and CRH-R2 (Fig. 4, A–C) receptor mRNAs
were present only in pregnant myometrium. Furthermore, the expression
of CRH-R1 and CRH-R1ß receptor mRNA appeared to be higher in the
pregnant human myometrium, although no direct statistical analysis of
the results could be employed due to the qualitative nature of the
methods used.
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, the
cells that showed actin-positive TRITC staining in their cytoplasm,
displayed a strong CRH-R-positive FITC staining on their cell membrane
(A and B). In addition, in a sequential tissue section,
immunofluorescence using vimentin/CRH receptor antibodies demonstrated
that cells positive for this fibroblast marker (TRITC stain) as well as
cells that were negative, i.e. smooth muscle cells, were
both positive for the CRH-R (FITC stain; Fig. 5, C and D), confirming
the presence of the CRH receptor on the cell membrane in both cell
types. The same studies were performed in sections from nonpregnant
uterus in which smooth muscle cells stained positive for CRH-R. These
sections, however, were almost devoid of fibroblasts, and we were
unable to demonstrate the presence of CRH-R in this cell type (data not
shown). No staining was detectable in sections in which the primary
anti-CRH receptor antibody was omitted or when sections were
preincubated with a specific CRH receptor antibody-blocking peptide
(data not shown).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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, 2, 1ß, and the variant C, were found in the
pregnant human myometrium at term before the onset of labor, whereas
only two subtypes, 1 and 1ß, were found in the nonpregnant
myometrium. This finding demonstrates for the first time that during
pregnancy there is an alteration in the pattern of CRH receptor subtype
expression. This original observation coupled with our previous studies
(4, 5, 10, 11) argue for a functional role for CRH and/or related
peptides in myometrial function. The biological role of placental CRH
is still uncertain, but it may modulate myometrial contractility, as it
is able to activate specific CRH receptors in the human myometrium.
These CRH receptors are capable of adopting a high affinity state
during the latter stages of pregnancy (4) when they become functionally
coupled to adenylate cyclase (10). Our hypothesis is further reinforced by the sequencing, cloning, and characterization of the various CRH receptor subtypes, all of which have been shown to activate adenylate cyclase in stable or transient transfection experiments (9). Full sequencing was essential in these studies because some of the primers under certain annealing conditions amplified DNA fragments of the correct size that on sequencing were found to be nonspecific. Furthermore, the presence of multiple CRH receptor subtypes in the human pregnant myometrium as identified in this study suggests distinct functional roles for each receptor during pregnancy and raises the possibility of multiple roles for CRH and/or related peptides. The intensity of the fluorescent signal detected after FISH analysis of pregnant myometrial tissue was greater than that in the nonpregnant myometrial tissues. This may indicate an increase in the level of expression of the CRH receptor mRNAs, but direct analysis of mRNA levels will be required to confirm this. Such analysis will require larger amounts of tissue than were available for this study. Furthermore, immunofluorescence studies demonstrated that the increase in CRH receptor expression in the pregnant biopsies may be due to the higher content of these biopsies in fibroblasts, as we identified CRH receptors in this type of cell. To our knowledge, this is the first time that the CRH receptor has been identified in fibroblasts. At present, the role of CRH or its receptor in these cells is unknown, but it may be involved in important changes occurring during pregnancy, such as cervical remodeling. Because of the lack of CRH-R-specific subtype antibodies, however, we were unable to identify which CRH-R subtypes are present in which cell type. Nevertheless, it appears that as pregnancy progresses there is a change in the range of CRH receptor subtypes. The functional consequence of this is unknown.
Of particular interest is the finding that the CRH-R2 receptor
subtype is present in human pregnant myometrium. To date the CRH-R2
receptor was thought to be confined solely to the central nervous
system (7). To our knowledge, this is the first report where the
CRH-R2 receptor has been identified in a tissue other than the
brain. As there are indications that another, as yet unidentified,
peptide (not CRH) is the native ligand for this CRH receptor subtype,
this raises the possibility that other signals may mediate the effects
of CRH-R2 on myometrial function. Our results also suggest that
CRH-R2 is only expressed in the pregnant, not in the nonpregnant,
smooth muscle cells of the human uterus. The lack of a CRH-R2-specific
antibody did not allow us to establish using immunofluorescence whether
this CRH-R subtype is also expressed in fibroblast cells of the
pregnant uterus. However, morphological studies using FISH suggested
that the CRH-R2 was expressed in both cell types.
Although the physiological role of CRH in myometrial function is not fully understood, the pattern of expression of the CRH receptor mRNA suggests that this receptor has a physiological role in the regulation of uterine function. Further studies are currently under way to identify the distribution of CRH receptor subtypes in uterine feto-maternal regions as well as to investigate the signals that regulate the pattern of expression of the CRH receptor subtypes.
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Footnotes |
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2 Coventry General Charities Ph.D student.
3 WPH Charitable Trust Chair of Medicine.
Received October 28, 1997.
Revised December 17, 1997.
Accepted April 14, 1998.
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References |
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