Mol. Hum. Reprod. Advance Access originally published online on March 25, 2004
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Molecular Human Reproduction, Vol. 10, No. 5, pp. 339-346, 2004
© European Society of Human Reproduction and Embryology 2004
Oxytocin receptor gene expression of estrogen-stimulated human myometrium in extracorporeally perfused non-pregnant uteri
1Department of Obstetrics and Gynaecology and 2 Department of Pathology, University of Bonn, School of Medicine, 53105 Bonn, Germany and 3Department of Obstetrics and Gynaecology, University of Bonn School of Medicine, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany. e-mail: Dr.OliverRichter{at}t-online.de
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Oxytocin (OT) and the oxytocin receptor (OTR) seem to be less important for uterine contractility-associated disorders of the non-pregnant uterus compared to the pregnant uterus. In the present study, we investigated the mutual dependence of OTR, OT and 17ß-estradiol (E2) with regard to the localization of OTR in the non-pregnant uterus. Utilizing our established model for extracorporeal perfusion of the human uterus, we perfused 15 human uteri for 27 h under physiological conditions without oestradiol (group A, n = 5) or with high E2 stimulation (group B, n = 5) followed by OT stimulation for both groups during the last 3 h of the experiment. Negative controls (n = 5) remained in perfusions for 27 h without any further hormone treatment. Gene expression of the myometrial OTR in both groups was compared using reverse transcriptase triple primer PCR. Stimulation with E2 and OT led to significantly higher OTR gene expression than stimulation with OT alone. We also showed that concentrations of OTR transcripts increase from the lower uterine segment to the uterine fundus. However, maximum OTR levels of the uterine fundus in group B did not reach concentrations of specimens of third trimester of pregnancy which were used as positive controls. We conclude that our experimental model simulates a situation similiarly to the stimulated non-pregnant uterus in the therapeutic concepts of assisted reproduction. The data presented demonstrate that the dynamics of OTR expression can be modulated by stimulation with E2 and OT, not only in the pregnant but also in the non-pregnant uterus.
Key words: Key words: estradiol/oxytocin/oxytocin receptor/oxytocin receptor gene expression/reverse transcriptase–triple primer–polymerase chain reaction
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Introduction |
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While the nonapeptide hormone OT plays an important role in many reproduction-related functions such as control of the estrous cycle length, follicle luteinization in the ovary and ovarian steroidogenesis, it is believed to have only minor significance for uterine contractility in the non-pregnant uterus (Bossmar et al., 1995; Fuchs et al., 1998; Gimpl and Fahrenholz, 2001). However, some investigators postulate dyscontractile disorders of the non-pregnant uterus to be receptor-mediated, and it has been found that both vasopressin and OT levels are increased in women with dysmenorrhoea, leading to intensified uterine activity and pain (Ekström et al., 1992; Akerlund et al., 1995; Noe et al., 1999).
In the uterine myometrial cell the estrogen receptor (ER), the progesterone receptor (PR) and the oxytocin receptor (OTR) are induced by estradiol stimulation (Soloff, 1975; Kano, 1982; Wathes and Hamon, 1993; Wathes et al. 1996; Adachi and Oku, 1995) and down-regulated by progesterone application (Fuchs et al., 1983; Sheldrick and Flint, 1985; Maggi et al., 1992; (Wathes and Hamon, 1993; Wathes et al. 1996; Ing and Tornesi, 1997; Robinson et al., 2001). Consistent with these findings, it has been shown that OTR concentrations are higher during the follicular phase of the cycle than in the luteal phase, presumably reflecting the positive and negative effects of estrogen and progesterone activity on receptor synthesis and degradation respectively (Fuchs et al., 1985; Lessey et al., 1988; Noe et al., 1999).
Previous investigations have shown that the contractility of human pregnant as well as non-pregnant myometrium induced by the nonapeptide hormone oxytocin is mediated through a high-affinity and low-capacity cell membrane-bound OTR (Kimura et al., 1992a, 1996; Maggi et al., 1992; Tahara et al., 2000). The human myometrial OTR is a 389 amino acid protein with a relative molecular mass of 42 819 kDA and has seven putative transmembrane domains, typical for G-protein-coupled receptors (Kimura et al., 1992b, 1993).
OTR of human myometrium, endometrium and ovary are encoded by mRNA with 4.4 kb, whereas OTR of the breast is 3.6 kb (Kimura et al., 1992b).
Myometrial sensitivity to oxytocin seems to be related to the induction of OTR which may be regulated by 17ß-estradiol (E2) and OT (Wathes and Hamon, 1993; Wathes et al. 1996; Wada et al., 1995). During pregnancy the concentration of uterine OTR increases 
6-fold by 13–17 weeks and 80-fold by term with a further increase in women during labour (Fuchs et al., 1982, 1984).
mRNA levels of OTR in human myometrium increase with gestational age to reach 100-fold at 32 weeks of pregnancy and 300-fold at term compared with the non-pregnant uterus (Kimura et al., 1996). Although data are controversial regarding the influence of ovarian steroids on OTR expression throughout the menstrual cycle, the sensitivity to oxytocin in the non-pregnant uterus is believed to be dependent on the stage of menstrual cycle, the time of exposure to the peptide, and the receptor density (Bossmar et al., 1995; Phaneuf et al., 1997, 2000; Quinones-Jenab et al., 1997; Einspanier et al., 1998; Kimura, 1998; Starbuck et al., 1998; Feng et al., 2000; Gimpl and Fahrenholz, 2001; Kitazawa et al., 2001).
Several investigators have pointed out the importance of in vivo studies for understanding physiological and pathophysiological conditions in the non-pregnant uterus.
Previous results have shown that extracorporeal perfusion of the human uterus under physiological conditions is a promising experimental system to investigate myometrial activities stimulated by ovarian hormones (Bulletti et al., 1986, 1988, 1993). Based on these experiences, we have established an extracorporeal perfusion system for the human uterus (Richter et al., 1998, 2000). The present study was designed to investigate the reactivity of myometrial OTR in the absence of E2 and OT (control group) or under high levels of E2 and increasing concentrations of OT (a condition similiar to the stimulated uterus in assisted reproduction treatment) by measuring OTR mRNA expression by RT–PCR methods.
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Surgical specimens and perfusion procedure
Fifteen uteri in the proliferative phase of the menstrual cycle were obtained by standard abdominal or vaginal hysterectomy for benign conditions, i.e. urine incontinence problems, descent of the uterus, dysmenorrhoea, from patients who had definitely completed their family planning. Exclusion criteria for the investigation were as follows: obesity, severe hypertension, diabetes mellitus, previous multiple abdominal surgeries, pelvic endometriosis and adenomyosis (>500 g), pelvic inflammatory disease, large fibroids and malignant diseases.
Oral contraceptives or HRT were discontinued 6–8 weeks prior to the operation, and hormone serum levels for each patient were evaluated on the day of operation for the assessment of E2, progesterone, LH and FSH. Ethical committee approval for the study protocol and written informed consent of the patients was given prior to the investigation.
As described previously (Richter et al., 1998, 2000), immediately after surgical removal of the respective uterus, both uterine arteries were cannulated with 14 G bulb-headed cannulas. Subsequent to bilateral flush perfusion with a modified heparinized Krebs–Henseleit bicarbonate buffer for removal of blood products, the organ perfusion with the recirculating perfusion system was started (Richter et al., 1998, 2000). Organ weights were always measured immediately before and after the experiment.
To investigate the effect of OT alone (group A) and of a combined treatment with E2 and OT (group B) on myometrial OTR expression of the non-pregnant uterus, we performed the following course of study: in the control group uteri were perfused for 27 h with the modified heparinized Krebs–Henseleit bicarbonate buffer without E2 treatment and without OT treatment (n = 5) (Figure 1a); in group A, uteri were perfused for 27 h with the modified heparinized Krebs–Henseleit bicarbonate buffer without E2 treatment but with OT treatment for the last 3 h of the experiment (n = 5) (Figure 1b); in group B, uteri were perfused for 27 h with the modified heparinized Krebs–Henseleit bicarbonate buffer with E2 treatment (1000 pg/min) throughout the perfusion period and with OT treatment for the last 3 h of the experiment (n = 5) (Figure 1c).
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Based on data of oxytocin, doses for induction and augmentation of labour (Muller et al., 1992) and the observation that uterine sensitivity to oxytocin is lower in early pregnancy than at term (Caldeyro-Barcia and Sereno, 1961), OT was added in group A and group B for the last 3 h of each experiment beginning with 8 mIU/min and increased by doubling the previous concentration every hour. All perfusion and monitoring proceedings were performed as previously described (Richter et al., 1998, 2000).
Immediately after surgical removal of the organ and until the end of the experiment, several myometrial biopsies from the fundus, the corpus and the lower uterine segment at 0, 12, 24, 25, 26 and 27 h of perfusion were excised >5 mm away from the serosal surface with a biopsy needle, washed in ice-cold normal saline, frozen in liquid nitrogen and stored at –80°C.
Positive controls were tissue fractions from patients in the 36th week of pregnancy who were not in labour and patients in the 40th week of pregnancy who were in labour who had to undergo Caesarean section.
The study protocol was approved by the Ethical Committee of the University of Bonn. Written informed consent was obtained from all patients.
RNA extraction and RT–TP–PCR
Quantification of oxytocin receptor expression.
Expression of OTR mRNA was quantified by RT–TP–PCR as described by Leygue et al. (1996). This technique consists of coamplification of truncated and wild-type cDNA in human tumour tissues using three primers in the PCR, where two primers anneal to individual sequences of the target cDNA but the third primer is common to both sequences. Thus, both targets compete for the common primer and the ratio of input cDNA. For quantification of OTR expression in individual specimens, we modified the approach described by Leygue et al. (1996) as described below.
Myometrial biopsies were taken as described above. Tissue fractions from pregnant women in the 36th and 40th week undergoing primary and secondary Caesarean section were used for plasmid cloning after RT–PCR with OTR specific primers.
RNA extraction.
After manual homogenization of the frozen tissue cylinders under sterile conditions, total RNA was extracted with a small scale extraction protocol (TrireagentTM; Sigma, USA) according to the manufacturer’s recommendations. The total RNA yield was quantified by spectrophotometry in a 70 µl microcuvette and the RNA concentration of the individual samples was adjusted to 150–250 µg/ml with DEPC-free sterile water. Samples were stored at –20°C until use.
Plasmid construction
OTR cDNA was amplified by RT–PCR with modified sequence-specific primers initially published by Helmer et al. (1995) and then cloned into plasmid pGEX-4T-1 (Amersham Pharmacia, Sweden). Reverse transcription of mRNA from uterine biopsies of term pregnant women was performed in a Gene-Amp 2400 PCR system (Perkin Elmer, Cetus Corp., USA) with a commercial kit (Ready-To-GoTM; Amersham Pharmacia Biotech, Sweden) which contains M-MuLV reverse transcriptase and Taq polymerase.
First strand cDNA was generated according to the manufacturer’s recommendations with pd (T)12–18 as the first strand primer at a reaction temperature of 42°C for 30 min.
In a second step, 20 pmol of sequence-specific primers for the human OTR gene were added to the reaction mixture and a PCR was run under the following conditions: an initial denaturation step at 95°C for 5 min was followed by 15 cycles of denaturation at 95°C for 40 s, annealing at 62°C for 40 s and chain elongation at 72°C for 1 min, followed by another 25 cycles with an annealing temperature of 70°C for 40 s under constant denaturation and synthesis conditions.
The primers used for amplification were modified from the publication by Helmer et al. (1995). An ECO RI restriction site plus an additional four bases were added to the 5' end of the 5' primer and an XHO restriction site plus an additional four nucleotides were added to the 3' end of the 3' primer to allow cloning of the resulting 248 bp fragment into the vector pGEX-4T-1.
Primer sequences were as follows: OTR-5ECO, 5'-GGCCGAATT CCTACCTGCTGCTGCTCATGTCC-3' (plasmid-5' primer; annealing temperature 82 °C); OTR-3XHO, 5'-ATATCTCGAGACCTCGCGCAGAGA GAAGATGT-3' (plasmid-3' primer; annealing temperature 75.9°C).
After identification and further culture of positive clones, the plasmid DNA was purified with the GFXTM Micro Plasmid Prep Kit (Amersham Pharmacia) according to the manufacturer’s recommendations. The average yield of plasmid DNA was 150 µg/ml. For further use in the triple primer PCR, the plasmid DNA was diluted 1:108 and a constant volume of 25 µl was added to the RT–PCR cocktail.
RT–TP–PCR
For quantification of human myometrial OTR expression, a RT–TP–PCR protocol (Leygue et al., 1996) was used with the following modifications. In a two-step approach, mRNA gained from the myometrial specimen was first transcribed into cDNA with the Ready-To-Go RT–PCR Kit as described above. In the second step, a TP–PCR was performed after addition of plasmid DNA containing a segment of the OTR sequence and the following three primers to the initial reaction cocktail: OTR 5'-primer: 5'-Cy5-CTACCTGCT GCTGCTCATGTCC-3' (OTR 5'-primer; annealing temperature 68.2°C); OTR 3'-primer: (5'-TAGCGTGATCCATGTGATGTAGG-3' (OTR 3'-primer; annealing temperature 65.8°C); OTR 3'-PLA: 5'-ATCGTCAGTCAG TCACGATGCG-3' (OTR 3'-primer, annealing temperature 69.7°C).
The OTR 5'-primer and OTR 3'-primer have been previously published (Helmer et al., 1995). The OTR 5'-primer binds to both the OTR and the plasmid sequence, whereas OTR 3'-primer is only specific for the OTR sequence and OTR 3'-PLA is specific for the plasmid sequence.
Thus, both targets (plasmid and OTR cDNA) compete for the common OTR 5'-primer and the ratio of RT–TP–PCR products obtained is directly related to the initial ratio of input cDNA. The OTR 5'-primer was labelled with the fluorescent residue 5-fluorocytosine (Cy5) to enable detection of the PCR products on an automated sequence detection system (ALF Automated Sequencer; Amersham Pharmacia). PCR conditions for the OTR-specific TP-PCR were as follows: an initial denaturation step at 95°C for 5 min was followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 63°C for 30 s and synthesis at 72°C for 30 s. PCR fragment sizes were 234 bp for the OTR and 187 bp for the plasmid.
Due to the very low concentrations of target cDNA used in the PCR set-up, amplification was still in the linear range after 40 amplification cycles.
Fragment analysis.
The fluorescently labelled PCR products were electrophoresed on a denaturing acrylamide/bisacrylamide gel with 0.6% TBE running buffer on an ALF automated sequencer. A volume of 5 µl of PCR product was diluted with an equal amount of running dye (Amersham Pharmacia), denatured at 98°C for 5 min and put on ice. Samples were then loaded on a 6% acrylamide/bisacrylamide gel (ratio 29:1) with a gel thickness of 0.5 mm and run with a sampling intervall of 2 s at 1500 V, 38 mA, 35 W and 56°C. Two fragmented standard ladders of 50–500 bp were run together with the samples.
Samples were analysed with the fragment manager software (Amersham Pharmacia) and the following parameters were evaluated for every PCR product: peak height, peak size and peak area. The OTR expression was measured as peak area of the specific OTR PCR product in relation to the peak area of the plasmid-specific PCR product which was set at 100%. For each sample, at least six independent PCR amplifications were performed to compensate for PCR-dependent variations.
Additionally, five independent gel runs were performed for every sample to compensate for variations between different gels. Results were corrected for PCR and gel-dependent variations.
Statistical analysis
All biomathematical evaluations were performed using the Stat View 5.0 statistical software (SAS Institute Inc., USA). Biochemical perfusion parameters were statistically evaluated using the Kruskal–Wallis one-way analysis of variance for non-parametric data as previously described (Richter et al., 2000).
For statistical evaluation of the OTR–TP–PCR results, we performed an analysis of variance (ANOVA) followed by Fisher’s protected least significance difference (PLSD) test and paired t-tests. P < 0.05 was considered significant.
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Results |
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Perfusions
All 15 patients were in a premenopausal proliferative status at the time of hysterectomy. In nine cases a contraceptive or hormone treatment with various estrogen–gestagen combinations was discontinued 6–8 weeks prior to the operation; six patients did not have any hormonal therapy. The median age was 38 years, range 31–46.
Hormone serum levels were as follows: median (minimal/maximum levels) E2: 107.4 (73.2/143.5) pg/ml; progesterone: 1.15 (0.61/1.45) ng/ml; LH: 6.9 (5.1/8.7) IU/l; FSH: 4.6 (3.9/5.7) IU/l.
Similiar to our previous results (Richter et al., 1998, 2000), all biochemical parameters (pH, pO2, pCO2, lactate, lactate dehydrogenase and creatine phosphokinase) were initially elevated due to preparation and cannulation time. In the further course of the perfusion they decreased significantly to physiological levels (for both groups 
= 0.5; P < 0.01) and remained stable as a sign of physiological oxygen consumption of the perfused organ (data not shown). Perfusions in both groups were maintained in physiological ranges with constant flow rates of 15–35 ml/min through each artery and pressure rates from 70–130 mmHg. There was no significant edema formation as evaluated by comparison of the organ weights before and after the perfusion (Table I), and examination by light microscopy showed intact tissue vitality and architecture throughout the entire perfusion period for all organs (Figure 3a, and b).
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There were no significant differences between group A and group B regarding hypoxia and cytolysis parameters respectively (data not shown).
RT–TP–PCR
From every specimen we performed six independent PCR amplifications and six independent gel runs to determine the intra-individual variability of each probe. The statistical evaluation using analysis of variance and Fisher’s protected least-significant difference test showed a homogeneous distribution of the PCR products for all samples of the respective biopsy time within the five uteri of the corresponding group (data not shown). The maximum intra-assay variability was 21%, the maximum inter-assay variability was 23%.
Controls
Throughout the perfusion period, the detected OTR transcripts showed no significant difference in all localizations: (fundus12h 4.27 ± 1.47 versus corpus12h 4.12 ± 1.21 versus lower uterine segment12h 4.15 ± 0.79; fundus24h 4.85 ± 1.37 versus corpus24h 4.21 ± 0.99 versus lower uterine segment24h 4.12 ± 0.78; fundus27h 4.66 ± 1.43 versus corpus27h 4.37 ± 1.09 versus lower uterine segment27h 4.04 ± 1.1) compared to OTR values of the respective biopsies taken before starting perfusion experiments (fundus0h 4.33 ± 1.1 versus corpus0h 4.02 ± 0.91 versus lower uterine segment0h 4.1 ± 0.93) (Figure 2a–c).
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Positive controls of pregnant myometrium showed OTR values of 52.45 ± 4.39 (36 weeks) and 68.30 ± 4.80 (40 weeks) (data not shown).
Group A
In group A during the first 24 h of perfusion, no relevant increase in PCR products was detected. The first significant changes of OTR expression in group A were in the fundus and corpus after 1 h of OT perfusion at 25 h (fundus-A0h 4.38 ± 1.44 versus fundus-A25h 5.64 ± 1.15, P < 0.005; corpus-A0h 4.31 ± 0.46 versus corpus-A25h 4.63 ± 0.63, P < 0.01) (Figure 2a and b).
Further during the course of the experiment we detected a significant increasing concentration of OTR in the biopsies of fundus-A (26 h: 9.07 ± 1.91; 27 h: 12.18 ± 2.95) and corpus-A (26 h: 6.47 ± 0.57; 27 h: 8.45 ± 0.75) (P < 0.0001) (Figure 2a and b). Compared to the control biopsies for the lower uterine segment-A0h (4.24 ± 0.42) the first significant change of OTR gene expression was found after 2 h of oxytocin stimulation in lower uterine segment-A26h (5.87 ± 0.55; P < 0.0001). Further stimulation with oxytocin led to a significant increase in OTR expression in all compared specimens of the lower uterine segment (P < 0.0001) (Figure 2c).
Analogous to the results within the respective localizations, the comparison between fundus-A, corpus-A and lower uterine segment-A showed no significant difference in the detected PCR products in the first 24 h of perfusion.
In the biopsies taken at 25 h of the continuing experiment, the difference between fundus-A (5.64 ± 1.15) and corpus-A (4.63 ± 0.63) was significant (P < 0.001). Further examination of OTR gene expression at 26 and 27 h for fundus-A versus corpus-A showed statistically significant values (P < 0.0001) (Figure 2a and b).
Statistical relevance between fundus-A and lower uterine segment-A was reached at 25 h of the perfusion experiment. The comparison of fundus-A25–27h (data see above) and lower uterine segment-A25–27h (25 h: 4.20 ± 0.53; 26 h: 5.87 ± 0.55; 27 h: 6.99 ± 0.63) showed significant differences (P < 0.0001) (Figure 2c).
Significant changes between corpus-A and lower uterine segment-A were found in the following specimens: corpus-A25h (4.63 ± 0.63) versus lower uterine segment-A25h (4.20 ± 0.53), P < 0.01; corpus-A26h (6.47 ± 0.58) versus lower uterine segment-A26h (5.87 ± 0.55), P = 0.001; corpus-A27h (8.45 ± 0.75) versus lower uterine segment-A27h (6.99 ± 0.63), P < 0.0001 (Figure 2b and c).
Group B
Compared to the control group E2 stimulation in group B led to a significant increase in OTR concentrations within the respective biopsies after only 12 h of perfusion for fundus-B12h (6.26 ± 2.92) versus corpus-B12h (5.47 ± 1.35) versus lower uterine segment-B12h (4.75 ± 0.89), P < 0.0001 (Figure 2a–c).
The maximum OTR gene expression was detected in fundus-B at 27 h (32.33 ± 6.37) (Figure 2a) and reached on average 62% (P < 0.0001) of the OTR density evaluated in the myometrium of the 36th week of pregnancy and 47% (P < 0.0001) of the 40th week of pregnancy. The comparison of the 36th and 40th weeks of pregnancy was significant (P <0.0001) (scale bars not shown).
Similiar to the results of the OTR expression in group A, increasing concentrations of OTR transcripts from the lower uterine segment to the fundus were noticed (Figure 2a–c). Comparing fundus-B and corpus-B, the first significant difference in OTR expression occurred after 24 h of perfusion fundus-B (24 h: 14.01 ± 4.30; 25 h: 19.65 ± 5.51; 26 h: 27.43 ± 5.93; 27 h: 32.33 ± 6.37) versus corpus-B (24 h: 9.16 ± 1.04; 25 h: 10.22 ± 1.70; 26 h: 18.41 ± 1.67; 27 h: 20.42 ± 3.10) (P < 0.0001) (Figure 2a and b).
The comparison between fundus-B and lower uterine segment-B showed statistically different OTR concentrations after 12 h of perfusion for fundus-B (12–27 h: see data above) versus lower uterine segment-B (12 h: 4.75 ± 0.84; 24 h: 5.30 ± 1.11; 25 h: 7.02 ± 1.26; 26 h: 9.10 ± 1.17; 27 h: 9.98 ± 1.34) (P < 0.0001) (Figure 2a and c).
The OTR density compared in corpus-B and lower uterine segment-B showed significant differences after a 24 h perfusion period (P < 0.0001) (Figure 2b and c).
Group A versus group B
After 12 h of perfusion we found a significant increase in the OTR gene expression in fundus-B (12 h: 6.26 ± 2.92) compared to fundus-A (12 h: 4.24 ± 1.59) (P < 0.001) (Figure 2a). Further during the course of the experiment, all estradiol-stimulated specimens of fundus of group B showed significantly higher OTR gene expression than myometrial tissue of group A at the respective time of biopsy (P < 0.0001) (Figure 2a). For corpus-A and corpus-B as well as for lower uterine segment-A and lower uterine segment-B we found no statistically significant difference of OTR gene expression between both groups during the first 24 h of perfusion (Figure 2b and c).
Biopsies taken at 24 h and later showed significant results for corpus-A (24 h: 4.26 ± 0.97; 25 h: 4.93 ± 1.13; 26 h: 6.47 ± 1.58; 27 h: 8.45 ± 1.35) versus corpus-B (24 h: 9.16 ± 1.04; 25 h: 10.22 ± 1.70; 26 h: 18.41 ± 1.67; 27 h: 20.42 ± 3.10) and lower uterine segment-A (24 h: 4.03 ± 0.92; 25 h: 4.20 ± 1.05; 26 h: 5.87 ± 1.55; 27 h: 6.99 ± 1.33) versus lower uterine segment-B (24 h: 5.30 ± 1.11; 25 h: 7.02 ± 1.26; 26 h: 9.10 ± 1.17; 27 h: 9.99 ± 1.34) (P < 0.0001) (Figure 2b and c).
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Discussion |
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In the present study we examined the gene expression of the oxytocin receptor by RT–TP–PCR in the human non-pregnant myometrium in relation to localization and estradiol and oxytocin stimulation using a previously established perfusion system for the human uterus (Richter et al., 1998, 2000).
The preservation of the human perfused uterus—as performed in the present study—has been biologically validated by extensive biochemical, light- and electron-microscopic examinations and was confirmed to be a new scientific approach, especially for mid-term and long-term perfusion experiments (Richter et al., 1998, 2000). Furthermore, experimental pharmacology to control uterine activities can easily be investigated, avoiding the risks of in vivo testing for the patient.
Regarding tissue viability and architecture in the present investigation, the examined hypoxia and cytolysis parameters were maintained in physiological ranges throughout the entire perfusion period, and the obtained perfusion data showed no significant differences between group A (without E2 stimulation) and group B (with E2 stimulation) respectively.
Contrary to its important role in many reproduction-related functions, such as control of the estrous cycle length, follicle luteinization in the ovary and ovarian steroidogenesis, OT is believed to have only subordinate significance for the uterine contractility in the non-pregnant uterus (Bossmar et al., 1995; Fuchs et al., 1998; Gimpl and Fahrenholz, 2001). Investigations by Akerlund et al. (1994, 1995) and Ekström et al. (1992) have shown that both vasopressin and oxytocin levels are increased in women with dysmenorrhoea, leading to intensified uterine activity and pain. Maggi et al. (1992) detected both vasopressin type V1a and oxytocin receptors in the myometrium of non-pregnant women. Further experiments showed that OTR mRNA is expressed in leiomyomas and myometrium of the non-pregnant uterus in higher concentrations in the luteal phase compared to the post-ovulatory phase of the estrous cycle (Lee et al., 1998).
With respect to the time-dependent modulation of OTR mRNA in human and rat myometrial cells by exposure to OT, controversial data have been reported (Engstrom et al., 1988; Adachi and Oku, 1995; Phaneuf et al., 1997, 2000; Quinones-Jenab et al., 1997). Long-term OT stimulation of human non-pregnant myometrial cells up to 48 h lead to OTR mRNA down-regulation including transcriptional suppression and destabilization of mRNA by RNA-binding proteins, but the total amount of receptor protein seemed to be unaffected (Phaneuf et al., 1997, 2000). Furthermore, OT treatment resulted in decreasing surface OTR concentrations of human and rat myometrium, whereas the binding affinity of OT to OTR was unaffected (Engstrom et al., 1988; Adachi and Oku, 1995). On the other hand, during a short period after additional stimulation with OT, the total OTR concentration remained unchanged (Adachi and Oku, 1995) and similiar experiments in rats found no such effects on endometrial and myometrial OTR mRNA after OT stimulation (Wathes et al., 1996).
Our results are in contrast to these findings. We showed that in group A (the group without E2 stimulation) oxytocin acts specifically on its own receptor and is able to increase significantly the OTR gene expression for all samples of fundus, corpus and lower uterine segment respectively (Figure 2a–c). These findings corroborate previous investigations which showed that oxytocin has a specific effect on its own receptor in the pregnant as well as in the non-pregnant uterus (Bossmar et al., 1994, 1995; Wathes et al., 1996). However, it may be speculated that in our experimental condition—if OT stimulation was continued up to 48 h—down-regulation of the myometrial OTR might be detected. Further experimental results seem to confirm this assumption, in particular at the post-transcriptional level (unpublished data).
Furthermore, we observed not only increasing OTR up-regulation within the respective compared locations, i.e. comparison of all fundus specimens, but also a significant increase in OTR gene expression from lower uterine segment to the fundus at the respective times of biopsy (Figure 2a–c).
Our findings to a great extent confirm previous results of Fuchs et al. (1984, 1985) except that we found maximum OTR expression in the fundal myometrium whereas the previously described distribution showed similiar or higher OTR-binding sites in the corpus, particularly in the pegnant uterus.
Function as well as physiological regulation of the OT/OTR system are proposed to be strongly steroid dependent (Soloff et al., 1975, 1983; Adachi and Oku, 1995; Larcher et al., 1995; Wathes et al., 1996; Lee et al., 1998; Gimpl and Fahrenholz, 2001). However, data are still controversial and some studies have shown that there is no relevant variation in the myometrial OTR density during the menstrual cycle. Therefore, it was suggested that OT and OTR are less important for the regulation of uterine activity in the non-pregnant uterus than in pregnant women, in particular at term (Bossmar et al., 1995; Engstrom et al., 1998).
Our data reveal that myometrial OTR mRNA expression in the pre-menopausal non-pregnant human uterus obtained in the proliferative phase of the cycle can be increased by stimulation with E2 and oxytocin. This effect was discovered by analysis of OTR gene expression at the transcriptional level using RT–TP–PCR. These results are in accordance with previous observations in ruminants at the protein level—describing significantly increased oxytocin high-affinity sites at estrus of ewes (Roberts et al., 1976)—and at the transcriptional level showing that OTR mRNA expression is regulated cyclically and that the highest expression could be detected at estrus and the lowest during the luteal phase (Robinson et al., 2001; Wathes and Hamon, 1993; Zingg et al., 1995).
In group B (the group with E2 stimulation), we could show an analogous but much earlier development of the increasing OTR gene expression in the respective localizations. Obviously, E2 has a stimulating effect on OTR expression as the first change in OTR up-regulation reached significant values between 12 and 24 h of stimulation, whereas the earliest effect in group A (without E2) occurred after 25 h.
Furthermore, comparing the respective specimens of group A and group B, the first significant difference in OTR gene expression became apparent after 12 h of perfusion in fundus specimens (Figure 2a). Statistically different OTR gene expression values between group A and group B in biopsies of corpus and lower uterine segment were reached at 24 h of the continuing experiment (Figure 2b and c).
On closer examination, OTR gene expression rises significantly more in the combined E2–OT-stimulated situation than under OT stimulation alone (Figure 2a–c). That is why we propose—even during a short period of OT stimulation—that E2 is able to intensify the self-up-regulating effect of oxytocin on its own receptor.
With respect to the effect of E2 on OTR expression, these data are in accordance with previous observations in the rat where the first significant changes were detected after 12 h (Soloff et al., 1975, 1983). Although loss of estrogen receptors in cultured human myometrial cells has been reported (Severino et al., 1996), experiments with human myometrial monolayer cultures demonstrated similiar effects, albeit E2-induced OTR up-regulation occurred between 24 and 72 h, depending on the E2 concentration (Adachi and Oku, 1995).
Previous investigations showed significantly increased human ER in the late proliferative phase of the cycle with a characteristic distribution throughout the uterine wall (Noe et al., 1999) and E2 was supposed to up-regulate both ER and PR in human fibroids and homologous myometrium respectively (Englund et al., 1998). However, as there has been reported a rapid loss of ER and PR in human cultured myometrium and leiomyoma cells, independent of the presence or absence of E2 and/or progesterone (Severino et al., 1996) and, on the other hand, explanted and cultured human endometrial cells showed spontanous up-regulation of the OTR (Sheldrick et al., 1993) an alternative pathway for the induction of OTR synthesis on the transcriptional level was suggested (Ivell et al., 1997).
Investigations by Bulletti et al. (1993) have shown that E2 is able to increase both the electrical and mechanical uterine activity, whereas progesterone has a correspondingly decreasing effect on the non-pregnant human uterus (Bulletti et al., 1993). Furthermore, progesterone leads to OTR inhibition by direct binding (Grazini et al. 1998).
The increasing myometrial contractility may be caused by rising OTR concentrations mediated via E2 and OT stimulation. In this context, our results may advance previous observations that during the follicular phase of the menstrual cycle uterine contractility is induced and regulated by the E2 released by the dominant follicle together with autocrine/paracrine effects subsequent to stimulation of OT and the OTR within the endometrial–subendometrial unit (Kunz et al., 1998). Furthermore, as demonstrated by Rezapour et al. (1996), our results suggest—similiar to women during labour—that individual myometrial sensitivity is an important determinant of the response to administered OT in humans (Rezapour et al., 1996).
The maximum OTR density was detected for both groups in the fundus uteri with decreasing OTR concentrations towards the lower uterine segment (Figure 2a–c). As the mechanism of the periodicity and coordination of the force produced by the uterine myometrium is not fully understood and—as it is known that there are no comparable specific conducting bundles to coordinate cyclic myometrial contractions such as the bundle of His and the Purkinje fibre network in the heart—it could be suggested that stimulated fundal myometrial cells in the case of high OTR expression may play the role of contractile pacemakers using gap junctions, which have been found to build the connecting element between the uterine smooth muscle cells (Garfield et al., 1977; Guyton, 1991). Investigations by Dorn et al. (1999) have shown that during assisted reproduction procedures, significantly increased serum oxytocin levels in the patients can be observed. Similiar to these clinical conditions, our experimental model also simulates a situation comparable to the stimulated non-pregnant uterus.
Taken together, these observations suggest that OT/OTR system may have much more influence on the non-pregnant uterus, in particular on the therapeutic concepts of assisted reproduction, than currently appreciated.
In conclusion, we have demonstrated that the OTR gene expression is up-regulated by E2 and OT in the non-pregnant myometrium using an experimental model of the extracorporeal perfusion of the human uterus and RT–TP–PCR method. Extracorporeal uterine perfusion, which to the best of our knowledge was used for the the first time to investigate the influence of E2 and OT on the OTR gene expression in the human uterus, represents an exciting scientific approach for the study of dyscontractile phenomena in gynaecology and reproductive medicine.
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Acknowledgements |
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This research was supported by the Deutsche Forschungsgemeinschaft (DFG), Gz RI 958/1-1 and the BONFOR-Forschungskommission, Gz 103/17.
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TopABSTRACTIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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Submitted on October 7, 2003; accepted on November 25, 2003.
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