Reduced expression of CRH receptor type 1 in upper segment human myometrium during labour

Paired upper and lower uterine segmental myometrial tissues from pregnant and nonpregnant women were collected in Navy General Hospital, the teaching hospital of Second Military Medical University, Beijing, China. Approval of this study was granted by human ethic committee of Navy General Hospital as well as human ethic committee of Second Military Medical University. Each patient signed approved informed consent form to participate in this study. Nonpregnant (NP) myometrium tissues were obtained from normal cycling patients (n = 8) undergoing hysterectomy for fibroids.

Pregnant myometrial tissues were collected at cesarean section from pregnant women at term gestation and were divided into two groups: (1) term no labour (TNL, 37–41 weeks, n = 10), (2) term labour (TL, 37–41 weeks, n = 9). Labour was defined as regular contractions (<5 min apart) plus membrane rupture and cervical dilation (>3 cm) with no augmentation. Indications for cesarean section included breech presentation, placenta previa, previous cesarean section, cephalopelvic disproportion, failure of labour to progress, fetal distress, or maternal request. None of the women included in this study had evidence of underlying disease (e.g. hypertension, diabetes, preeclampsia, intrauterine growth restriction, etc.). LS uterine samples were collected from the upper margin of the LS uterine incision, while US uterine samples were taken from just below fundus. The nonpregnant tissues were taken from the normal part of uterus without fibrosis contamination. Collected samples were placed in phosphate-buffered saline (PBS) on ice and transported to the laboratory. Myometrial tissues were separated from any serosal or decidual components and then rinsed in PBS, frozen immediately in liquid nitrogen and stored at -80 C for Western blot and PCR analysis. Parts of the tissues were also placed in 10% phosphate buffered formalin for immunohistochemical analysis.

Paraffin sections (5 μm) were cut, rehydrated and microwaved in citric acid buffer to retrieve antigens. After inhibition of endogenous peroxidases with 3% H₂O₂, nonspecific antibody binding was blocked with 10% rabbit serum for 30 min. Serial sections were then incubated with specific antibodies against human CRH-R1 (sc-12381, Santa Cruz Biotechnology, Santa Cruz, California, USA) and CRH-R2 (sc-20550, Santa Cruz) (1:500) overnight at 4°C. The CRH-R1 antibody is directed against an epitope between amino acid positions 81 and 109 of CRH-R1 of human origin, where no sequence homology exists to CRH-R2. The CRH-R2 antibody was raised against a peptide mapping near the C-terminus of CRH-R2 of human origin. Mouse antihuman smooth muscle α-actin-specific monoclonal antibody (Dako Inc. Carpinteria, California) was used to stained the muscle cells. The bound antibodies were detected with the biotin-streptavidin-peroxidase system (UltraSensitive-SP-kit, MaiXin Biotechnology, Fuzhou, China) and diaminobenzidine (Sigma) was used as chromogen. Counterstaining was performed with hemalum. Negative controls were performed by substituting primary antibody with a normal serum or IgG in same dilution. To confirm the specificity of primary antibody, preabsorption of the primary antibody with a tenfold excess of the blocking peptides sc-12381P or sc-20550P (Santa Cruz) was performed.

Approximately 70 mg of human myometrial tissue was homogenized in ice-cold lysis buffer consisting of 60 mM Tris-HCl, 2% sodium dodecyl sulfate (SDS), 10% sucrose, 2 mM phenylmethylsulfonyl fluoride (Merck, Darmstadt, Germany), 1 mM sodium orthovanadate (Sigma-Aldrich), 10 μg/ml aprotinin (Bayer, Leverkusen, Germany). Lysates were then quickly sonified in ice bath, boiled 5 min at 95 C, and stored at -80 C until used. Protein concentrations were measured using a modified Bradford assay and samples were diluted in sample buffer (250 mM Tris-HCl (pH 6.8), containing 4% SDS, 10% glycerol, 2% β-mercaptoethanol, and 0.002% bromophenol blue) and boiled for a further 5 min. Samples (50 μg) were separated on an SDS-8% polyacrylamide gel, and the proteins were electrophoretically transferred to a nitrocellulose membrane at 300 mA for 1.5 h in a transfer buffer containing 20 mM Tris, 150 mM glycine, and 20% methanol. The membrane was then blocked in TBS containing 0.1% Tween-20(TBST) and 5% dried milk powder (wt/vol) for 2 h at room temperature. After three washes with TBST, the nitrocellulose membranes were incubated with primary antibody for CRH-R1 or CRH-R2 (1:500) at 4°C overnight. After another three washes with TBST, the membranes were incubated with a secondary horseradish peroxidase-conjugated IgG (1:1000) for 1 h at room temperature and further washed for 30 min with TBST. Immunoreactive proteins were visualized using the enhanced chemiluminescence Western blotting detection system (Santa Cruz). The light-emitting bands were detected with X-ray film. The resulting band intensities were quantitated using an image scanning densitometer (Furi Technology, Shanghai, China). To control sampling errors, the ratio of band intensities to β-actin was obtained to quantify the relative protein expression level.

Total RNA was extracted from the samples of myometrium by a method based on Chomczynski and Sacchi [29]. Briefly, frozen tissue samples were powdered under liquid nitrogen and homogenized in 1 ml of TRIzol reagent (Invitrogen, Grand Island, New York, USA). Subsequent extraction of total RNA from the tissues was conducted according to the protocol provided by the manufacturer. All RNA samples were treated with deoxyribonuclease I (Promega, Biotech. Co Ltd, Beijing, China). The purity and integrity of the RNA was checked spectroscopically and by gel electrophoresis. Two microgram of total RNA was reverse transcribed using the SuperScript^® first-strand synthesis system (Invitrogen) and stored at -20°C.

The specific primers for the amplification of the CRH-R2 variants and CRH-R1α/CRH-R1β were used as described previously [22, 23, 30]. The nucleotide sequences of the primers were as followings: (1) CRH-R2α, sense 5'-GAGCTGCTCTTGGACGGC-3', antisense 5'-GACAAGGGCGATGCGGTA-3'; (2) CRH-R2β, sense 5'-CCCTCACCAACCTCTCAGGTCC-3', antisense 5'-CAGGTCATACTTCCTCTGCTTGTC-3'; (3) CRH-R2γ, sense 5'-CTCAAGCAATCTGCCTACCT-3', antisense 5'-GGCTCACACTGTGAGTAGTT-3'; (4) CRHR1 α/β, sense 5'-GGCAGCTAGTGGTTCGGCC-3', antisense 5'-TCGCAGGCACCGGATGCTC-3'. PCR reaction solution consisted of 2.0 μl diluted cDNA, 0.4 μmol/L of each paired primers, 2.5 mmol/L Mg²⁺, 250 μmol/L deoxynucleotide triphosphates, 1 U Taq DNA polymerase (Qiagen, Beijing, China), and 1× PCR buffer. PCR reaction was set at 94 C (45 s), 58 C (45 s), 72 C (1 min) in a total 40–80 cycles with a final extension step at 72 C for 10 min.

Primers for nested PCR were previously described [21‐23], and were designed to distinguish the different variants of CRH-R1. The nucleotide sequences of the first round primers were: exons 2–7, sense 5'-TCCGTCTCGTCAAGGCCCTTC-3', antisense 5'-GGCTCATGGTTAGCTGGACCAC-3'; exons 9–14, sense 5'-CCATTGGGAAGCTGTACTACGAC-3', antisense 5'-GCTTGATGCTGTGAAAGCTGACAC-3'. The nucleotide sequences of the second round primers were: exons 2–7, sense 5'-TGTCCCTGGCCAGCAACATCTC-3', antisense 5'-AGTGGATGATGTTTCGCAGGCAC-3';exons 9–14, sense 5'-GGGTGTACACCGACTACATCTAC-3', antisense 5'-TCTTCCGGATGGCAGAACGGAC-3'. RT-products (cDNA) from tissues were used as template for the fist round of PCR. After 40 cycles of amplification, 2 μL of the reaction mixture was used for additional 40-cycle amplification. The primers used in this second round of amplification were internal to the first set of primers.

Ten microliters of the reaction mixture were subsequently electrophoresed on a 1.5% agarose gel and visualized by ethidium bromide, using a 100 bp DNA ladder (Invitrogen) to estimate the band sizes. As a negative control for all of the reactions, distilled water was used in place of cDNA. The identity of the PCR products was confirmed by sequencing. Sequence data were analyzed using Blast nucleic acid database searches from the National Centre for Biotechnology Information (NCBI).

Protein expression levels of CRH-R1 and CRH-R2 were determined by densitometric analysis (Furi Technology, Shanghai, China). Peak count values were expressed as densitometric units. The data are presented as mean ± SEM. All data were tested for homogeneity of variance by Bartlett's test. The results indicated that the data were normally distributed. Student's t-test was used in the analysis of these data. One-way ANOVA with Student-Newman-Keuls was used for multiple comparisons. A P value < 0.05 was considered statistically significant.

Both CRH-R1 and CRH-R2 were identified in nonpregnant (Fig. 1) and pregnant (Fig. 2) myometrium. Localization of CRH-R1 and CRH-R2 showed that these receptors were highly expressed in the myometrial smooth muscle in US and LS (Fig. 1A–C and Fig. 2A, B and 2D–F arrowhead). Positive staining of CRH-R1 and CRH-R2 was also seen in vascular smooth muscle cells (Fig. 1D and Fig. 2C arrowheads). Staining in the US and LS was similar in each of groups and no dramatic changes in overall staining intensity or localization were observed with pregnancy or with labour (Fig. 2). CRH-R1 and CRH-R2 staining was eliminated when antibody was pre-absorbed by synthetic peptide (Fig. 1E and 1F, respectively).

As expected, Western blot analysis recognized a protein band of approximately 55 KDa corresponding to CRH-R1 in human myometrium (Fig 3A). A very faint band of about 47–50 KDa was also observed (Fig 3A). A single band of about 55 KDa corresponding to CRH-R2 was identified in human myometrium (Fig 3A). Preabsorption with corresponding peptides indicated the specificity of bands (c&d columns of Fig. 3A and 3B).

With US myometrium, the protein level of CRH-R1 was down-regulated in pregnancy (NP versus TNL, P <0.05, Fig 4). It was further decreased at the time of labour (TL versus TNL, P < 0.05, Fig 4). With LS myometrium, although there was a trend of decreased CRH-R1 expression with pregnancy, the difference failed to reach significance (Fig 4, NP versus TNL, NP versus TL). No significant difference in CRH-R1 levels between TNL and TL group was observed.

For CRH-R2 levels, no significant changes in associated with pregnancy or labour were found in US and LS myometrium (Fig. 5).

There was no difference in CRH-R1 expression between US and LS myometrim in NP and TNL samples. Interestingly, in TL samples, the LS myometrium expressed significantly more CRH-R1 when compared with US, in part due to the significant reduction in CRH-R1 expression in the US at the labour onset (Fig 4). No significant difference in CRH-R2 expression between US and LS myometrim was found across all groups (Fig 5).

RT-PCR and nested PCR analysis showed that CRH-R1α,-R1β,-R1c, -R1e,-R1f and -R1g were identified in nonpregnant as well as pregnant US and LS samples (Fig 6B and 6C). Other CRH-R1 variants were undetected. CRH-R1α and -R1β were detected in all biopsies from pregnant and nonpregnant women, whereas CRH-R1c, -R1e,-R1f and -R1g were not detected in all biopsies. The detection rates of CRH-R1c, -R1e,-R1f and -R1g was various in NP, TNL and TL groups. For instance, within US, CRH-R1c was identified in 1/9TL and 3/10 TNL samples. CRH-R1e was detected in 2/9 TL and 3/10 TNL biopsies. CRH-R1f and -R1g were detected in all TL tissues and 4/10 TNL tissues. Within LS, 3/10 TNL and 4/9 TL tissues for CRH-R1c, 3/10 TNL and 5/9 TL tissues for CRH-R1e, 4/10 TNL and 6/9 TL samples for CRH-R1f, and 3/10 TNL and 5/9 TL tissues for CRH-R1g were identified.

CRH-R2 has three variants, termed CRH-R2α, CRH-R2β and CRH-R2γ [20]. PCR analysis using specific primers for these CRH-R2 variants resulted in the detection of CRH-R2α in all of the pregnant myometrial biopsies and CRH-R2β in all of nonpregnant tissues (Fig 7). We are unable to detected CRH-R2γ (data not shown).