Molecular Human Reproduction, Vol. 5, No. 11, 1048-1054,
November 1999
© 1999 European Society of Human Reproduction and Embryology
Molecular events in the uterus |
Nitric oxide synthase expression and steroid regulation in the uterus of women with menorrhagia
1 Cell and Molecular Development Group, Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, and 2 Cardiff Assisted Reproduction Unit, University Hospital of Wales, Heath Park, Cardiff CF4 4XW, UK
Abstract
Menorrhagia (excessive menstrual bleeding) is a common clinical problem of unknown aetiology. The free-radical and vasodilator nitric oxide (NO) relaxes the myometrial smooth muscle and is a strong candidate for the cause of excessive blood loss in menorrhagic patients. The aim of this study was to measure NO production in women with and without menorrhagia to detect nitric oxide synthase (NOS) isoforms in uterine cells and to investigate any steroid effects on myometrial NOS expression. We showed for the first time that menorrhagic endometrium produces significantly higher amounts of NOx (the sum of NO2– and NO3–) than control endometrium (P < 0.01). Inducible NOS (iNOS) protein was detected by immunoblotting in endometrial and myometrial tissue extracts. Quantitative reverse transcription–polymerase chain reaction (RT–PCR) experiments revealed an induction of myometrial smooth muscle endothelial NOS (eNOS) expression by progesterone and 17ß-oestradiol, while myometrial iNOS expression was unaffected by steroid hormones. These results are consistent with the hypothesis that NO plays a role in excessive menstrual bleeding and provide the first evidence on steroid regulation of eNOS in the human non-pregnant uterus.
17-oestradiol/menorrhagia/nitric oxide/progesterone
Introduction
Menorrhagia, defined as excessive menstrual blood loss (Hallberg et al., 1966
), remains a challenge at the therapeutic and research level. In the UK, 31% of reproductive age women suffer from menorrhagia and 80–90% of these women receive unnecessary and ineffective treatment (Rees, 1991). More than 80% of menorrhagic women do not have an organic disease, such as uterine tumours (polyps, fibroids, carcinomas), infection or pregnancy.
Recent work has shown that blood clotting disorders were responsible for a proportion of menorrhagia cases, but not for the majority of them (Kadir et al., 1998). Much attention has been focused on the uterus itself, and it seems that locally derived factors and vascular changes control the amount of blood loss during menstruation. The correlation between uterine artery pulsatility index and amount of menstrual blood loss, suggests that vascular factors may be involved in the pathogenesis of menorrhagia (Hurskainen et al., 1999). Previous studies have also shown increased concentrations of prostaglandins F2
and E2 in menstrual fluid of women with menorrhagia (Rees et al., 1984b), and increased prostaglandin E2 receptors in myometrium, correlated with menstrual blood loss (Cameron et al., 1988).
The free radical gas nitric oxide (NO) is produced after catalytic oxidation of L-arginine by the enzyme nitric oxide synthase (NOS). NOS occurs in three distinct isoforms: two constitutive, Ca2+-dependent, isoforms endothelial NOS (eNOS) and neuronal NOS (nNOS), and the Ca2+-independent inducible NOS (iNOS) (Moncada et al., 1991). nNOS (NOS I) was first identified in neuronal tissues and is expressed constitutively, like eNOS (NOS III) which was first identified as a constitutive enzyme in vascular endothelial cells. iNOS (NOS II), first described in macrophages, can be induced by cytokines or bacterial lipopolysaccharides (LPS) (Fosterman et al., 1991). NO action has been associated with platelet aggregation, relaxation of smooth muscles in the gastrointestinal and respiratory tract, neurotransmission and immunoactivation. Such regulatory functions of NO are mediated via cyclic guanosine monophosphate (cGMP) (Moncada et al., 1991).
NO production has been assayed in the pregnant rat uterus, by measuring its oxidation products nitrite (NO2–) and nitrate (NO3–), and an L-arginine-NO-cGMP pathway was found to exist throughout pregnancy (Yallampalli and Garfield, 1993; Yallampalli et al., 1993; Jaing et al., 1996). In the rat and human myometrium, an L-arginine-NO system has an important role in inhibiting uterine contractility and possibly, maintaining pregnancy (Izumi et al., 1993). However, NOS expression decreases at term, when myometrial contractions lead to labour (Weiner et al., 1994b; Buhimschi et al., 1996; Dong et al., 1996; Bansal et al., 1997; Riemer et al., 1997; Norman et al., 1999). Various studies have suggested a role of NO in regulation of menstrual blood loss (Norman, 1996; Norman and Cameron, 1996; Telfer et al., 1997), and more recent work showed that NOS activity is increased during menstruation, suggesting a role of NO in menstrual blood release and in endometrial apoptosis (Tschugguel et al., 1999).
As well as affecting vasodilation by relaxing the vascular smooth muscle, uterine-derived NO appears to act as a suppressor of smooth muscle contractility, by maintaining uterine quiescence during pregnancy and relaxing the non-pregnant human myometrium (Cameron and Campbell, 1998).
Ca2+-dependent isoforms eNOS and nNOS were previously detected in human pregnant and non-pregnant endometrium and myometrium, while iNOS was detected in the pregnant and non-pregnant endometrium and pregnant myometrium (Bansal et al., 1997; Telfer et al., 1997; Norman et al., 1999). In view of the suggested roles of NO in pregnancy and menstruation, it is of interest to study eNOS and iNOS expression. eNOS is a known vasodilator of myometrial vasculature and neighbouring smooth muscle cells (Tschugguel et al., 1997) and iNOS was reported to have a steroid-mediated action in the pregnant rat uterus (Ali et al., 1997).
Data on the steroid regulation of NOS isoforms in the human non-pregnant uterus is still unclear. Ca2+-dependent NOS isoforms were reported to be induced by oestrogens in pregnant guinea pigs (Weiner et al., 1994a) and in non-pregnant sheep (Figueroa and Massmann, 1995). A progesterone-, but not 17ß-oestradiol-mediated regulation of vascular adaptations by NO was reported during pregnancy in rats (Buhimschi et al., 1995). Progesterone was also suggested to be the steroid hormone with the major steroid action on vascular tension during pregnancy (Liao et al., 1996).
Inducible NOS was found to play a role in initiation of labour and cervical ripening in rats and progesterone was found to differentially regulate iNOS in the rat cervix and uterus (Ali et al., 1997). Recently, a synergistic role of NO and progesterone during the establishment of pregnancy in the rat was demonstrated (Chwalisz et al., 1999). However, other workers (Telfer et al., 1997) observed neither a role of exogenous progestagens on human uterine NOS nor any significance of the phase of the menstrual cycle on the protein expression of NOS isoforms.
The aetiology of menorrhagia, however, remains largely unknown. We introduce the hypothesis that uterine cells (endometrial and/or myometrial) of women suffering from menorrhagia demonstrate an increased activity of NOS, a consequence of which is overproduction of NO, leading to dilatation, congestion of uterine vessels and excessive amount of menstrual blood loss. In addition to the main hypothesis we have also assessed the role of sex steroids in regulating NOS gene expression in the non-pregnant uterus, in an attempt to better understand the mechanisms of non-pregnant myometrial activity.
Materials and methods
Subjects
All tissue samples were collected from women undergoing a gynaecological operation at Women's Hospital, Walsgrave Hospitals' NHS Trust, Coventry, UK. The study was approved by the local research ethics committee, and informed consent was obtained from each patient prior to operation. Two groups of women participated in the study: (i) control group; 18 women (age 39–44 years), undergoing laparoscopy for sterilization and (ii) study group; 20 women (age 35–48 years) undergoing a hysterectomy for menorrhagia, under the diagnosis of dysfunctional uterine bleeding.
All women recruited to the study were pre-menopausal, with normal menstrual cycles (25–35 days), had not been exposed to steroid treatment for three months prior to operation, did not have either an intrauterine contraceptive device (IUD) in situ or evidence of uterine pathology, such as fibroids or polyps. Myometrial biopsies were taken from the upper third of the uterine body ~5 mm away from endometrial or serosal surfaces, immediately after hysterectomy. Endometrial biopsies were obtained by sharp dissection of uterine tissue. Myometrial samples were either snap-frozen in liquid nitrogen and stored at –70°C until use, or were collected in ice-cold Hanks' balanced salt solution (HBSS, Sigma, Poole, UK) containing penicillin G (10 000 IU/ml) (Sigma) and streptomycin (7610 IU/ml) (Sigma), for enzymatic dispersion and cell culture. For the control group, endometrial biopsies were obtained from either the fundus, or the upper third of the uterine body, using a Sharman's Curette (Dans Surgical Ltd, Sheffield, UK). The biopsies were snap-frozen in liquid nitrogen and stored at –70°C until further use.
NOx assays
Endometrial biopsies were collected from controls (n = 18) and from menorrhagic patients (n = 20). The chronological dating (based on the last menstrual period) of the biopsies is shown on Figure 1a. Frozen endometrial samples were weighed and homogenized by an Ystral homogenizer (7801, Dottingen, setting 6) at 0°C in 20 mmol/l Tris–HCl (pH 7.4), 1 mmol/l EDTA, 50 mmol/l NaCl (BDH, Poole, UK), 2 µg/ml aprotinin (Sigma), 1% phenylmethylsulphonyl fluoride (PMSF; BDH). Two sequential centrifugations were carried out at 13 000 rpm for 5 min at 4°C, in order to separate the cytosolic fraction (supernatant) from the membranous fraction (pellet). The latter was discarded.
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For NOx determination, a manual spectrophotometric version of the Griess reaction was carried out according to previously published protocols (Tracey, 1992
). Samples were incubated at 37°C for 15 min in a solution containing 0.1 IU/ml nitrate reductase (Sigma), 50 µmol/l NADPH (Roche Boehringer Mannheim, Mannheim, Germany) and 5 µmol/l FAD (Roche Boehringer Mannheim). Following nitrate reduction, NADPH was removed by incubation at 37°C for 5 min after addition of lactic dehydrogenase (LDH) (10 IU/ml) (Roche Boehringer Manheim) and sodium pyruvate (10 mmol/l) (BDH). The samples were then cooled at 4°C for 10 min and diazotization was performed by adding sulphanilamide (p-aminobenzene-sulphonamide; Sigma) at a final concentration of 1 mmol/l in 0.1 N HCl. Centrifugation followed at 4000 rpm for 15 min, and 150 µl of each of the supernatants were transferred to a corresponding well of a 96-well enzyme-linked immunosorbent assay (ELISA) plate. The absorbance of the supernatants was measured at 540 nm and was corrected for the mean absorbance of a column of blank wells containing homogenization buffer. N-(1-naphthyl) ethylenediamine (NEDA) (Sigma) was added to each well at a final concentration of 1 mmol/l, and after a 10 min incubation at room temperature, absorbance readings at 540 nm were obtained for a second time. The assay was calibrated against a series of reference standard dilutions, each one containing sodium nitrite (NaNO2) (BDH) and sodium nitrate (NaNO3) (Sigma) (at concentrations of 0–10 µmol/l). Absorbance values before addition of NEDA were subtracted from the second absorbance readings.
Immunoblotting
Snap-frozen endometrial and myometrial tissue was homogenized and cytosolic fractions were obtained as already described. For detection of iNOS, sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) was carried out. 80 µg of cytosolic protein were diluted 1:1 v:v with electrophoresis SDS sample buffer (125 mmol/l Tris–HCl, pH 6.8, 20% glycerol, 2% SDS, 0.003% Bromophenol Blue and 5% ß-mercaptoethanol) (BDH) and were reduced by heating at 95°C for 5 min. Positive control aliquots (Transduction Labs, Lexington, KY, USA) of 5 µl mouse macrophage lysate samples, stimulated by interferon (IFN)-
and LPS according to previous protocols (Xie et al., 1992), were also loaded. After SDS–PAGE and electrophoretic transfer to a nitrocellulose membrane (Hybond-C; Pharmacia Amersham Life Science, Little Chalfont, UK), non-specific sites were blocked overnight at 4°C, by 5% non-fat dry milk in Tris-buffered saline (TBS; 10 mmol/l Tris pH 7.5, 100 mmol/l NaCl) containing 0.01% Tween 20 (Sigma). Incubation with a mouse, anti-human iNOS antibody (Transduction Labs, 1:1000 dilution in TBS/Tween 20), raised against amino acids 961–1144 at the C-terminus of human iNOS, was carried out for 1 h at room temperature. The blot was then washed in TBS (containing 0.01% Tween 20), four times for 5 min each. Incubation with the secondary antibody, a horseradish peroxidase conjugated goat anti-mouse immunoglobulin G (IgG, Sigma; 1:300 dilution in TBS/Tween 20) followed for 45 min at room temperature. For detection of iNOS the membrane was subjected to enhanced chemiluminescence using the ECL detection reagents (Pharmacia Amersham) and then exposed to X-ray film (Kodak).
Myocyte culture
Myometrial biopsies, weighing ~3 g were collected from women undergoing hysterectomy for menorrhagia. Primary myocyte cultures were prepared as previously described (Phaneuf et al., 1993). Smooth muscle myometrial tissue was incubated at 37°C, 5% CO2 for 2 h with gentle shaking/stirring, in HBSS containing 2.5 mmol/l CaCl2, 0.9 mmol/l MgCl2 and 10 mg/ml (IU) dispase II (Roche Boehringer Mannheim). The softened pieces of myometrium were washed in Ca2+/Mg2+-free HBSS (Gibco BRL, Life Technologies, Paisley, UK) and the cell dispersion was obtained in the same solution containing 300 IU/ml collagenase type 1A (Sigma), 2 IU/ml elastase (Sigma), 30 IU/ml DNase I type IV (Sigma) and 1 mg/ml fatty acid-free bovine serum albumin (FAF–BSA) (Sigma), after incubation at 37°C, 5% CO2, for 2 h, as above. Tissue debris was removed by filtration through a 250 µm-diameter sterile sieve (Lockertex, Warrington, UK) and by washing three times with HBSS. Myocytes were resuspended in Dulbecco's modified Eagle's medium (DMEM) (Sigma), containing 10% fetal calf serum (Gibco BRL), 0.2% L-glutamine, 10 000 IU/ml penicillin G (Sigma) and 7610 IU/ml streptomycin (Sigma), supplemented by one of the following: 5 µmol/l progesterone (Sigma); 5 µmol/l RU 486 (mifepristone) (kindly donated by Prof. E.-E.Baulieu, INSERM, Paris, France); 5 µmol/l progesterone + 10 µmol/l RU 486; 5 µM 17ß-oestradiol (Sigma) + 5 µmol/l progesterone; 5 µmol/l 17ß-oestradiol. A `no supplement' culture served as a negative control. Myometrial cells were plated into 25 cm2 culture flasks (Nunc, Life Technologies, Paisley, UK), at a density of 0.5–2x104 cells/cm2 and stored at 37°C in humidified atmosphere (95% air and 5% CO2) for 72 h.
Cultured cells were identified as myometrial smooth muscle cells after performing immunocytochemistry using an anti-human smooth muscle -actin antibody (Gangula et al., 1997). Myocyte monolayers, grown on 2-chamber glass slides (Falcon, Becton Dickinson, Oxford, UK), were fixed in acetone:methanol (50:50), for 5 min before incubation with an anti-human -actin (Sigma), specific for smooth muscle cells (Roholl et al., 1990). Cells were then stained with a fluorescent tetramethyl-rhodamine isothiocyanate (TRITC)-conjugated IgG antibody (Sigma). Further staining with the DNA-specific dye 4,6-diamino-2-phenylindole (DAPI) (Sigma) allowed visualization of the cell nuclei.
Quantitative reverse–transcriptase polymerase chain reaction (RT–PCR)
Total ribonucleic acid (RNA) was extracted from myocyte monolayers originating from a menorrhagic patient, by solvent extraction using the ULTRASPEC reagent (Biotecx Laboratories, Houston, TX, USA), according to manufacturer's instructions. RNA was reverse transcribed into cDNA for use as the template for polymerase chain reaction (PCR) by using 2 µg total RNA samples and 5 IU/µl Maloney murine leukaemia virus (MMLV) RNase H reverse transcriptase (Superscript II) (Gibco BRL).
PCR was carried out with specific primers (Gibco BRL) for human eNOS; 5'-CAG TGT CCA ACA TGC TGC TGG AAA TTG-3' (sense) and 5'-TAA AGG TCT TCT TCC TGG TGA TGC-3' (antisense), (Marsden et al., 1993; Weiner et al., 1994c). Amplification for iNOS was carried out with primers 5'-GGA ATT CAC TCA GCT GTG CAT CG-3' (sense) and 5'-GTT TCC AGG CCC ATT CTC CTG C-3' (antisense) (Chartrain et al., 1994), as described previously (Telfer et al., 1997). For glyceraldehyde-3-phosphate dehydrogenase (GAPDH) the following pair of primers (Gibco BRL) was used; 5'-ACC ACA GTC CAT GCC ATC AC-3' (sense) and 5'-TCC ACC ACC CTG TTG CTG TA-3' (antisense), according to other workers (Ercolani et al., 1988). 1 µl of the generated cDNA reaction was then used in the amplification reaction. PCR was performed in a 25 µl final volume using 10 µCi/µl [32P]-labelled -dGTP and 5 IU/µl Taq DNA polymerase (Gibco BRL).
Amplifications were carried out as follows: for eNOS, 35 cycles of 30 s at 94°C, 1 min at 58°C and 1 min at 72°C. For iNOS, an initial step at 94°C for 2 min, followed by 35 cycles of 1 min at 94°C, 1 min at 55°C, 1 min at 72°C and a final step of 1 cycle of heating at 72°C for 10 min. Finally, for GAPDH, RT–PCR reactions were performed as follows: one cycle of denaturing at 94°C for 4 min, 1 min at 60°C and 68°C extension for 1 min, followed by 28 cycles of 30 s at 94°C, 30 s at 60°C and 2 min at 68°C. A final cycle of heating at 68°C for 5 min completed the amplification reaction for GAPDH. PCR products were added to 5 µl of denaturing stop solution (95% formamide, 20 mmol/l EDTA, 0.05% Bromophenol Blue, 0.05% xylene cyanol FF) (Pharmacia Amersham Life Sciences), denatured at 95°C for 5 min and separated by urea/acrylamide gel electrophoresis. The dried gel was either exposed to a Kodak X-ray film or, alternatively, to a phosphorimager screen (Molecular Dynamics, Pharmacia Amersham) for quantification purposes. Each product's peak was estimated by using the Microsoft Windows ImageQuant (IQ) program after scanning of the screen.
Cloning and sequencing of PCR products
To confirm the specificity of the RT–PCR products, the latter were electrophoresed non-radioactively, purified by using the QIAquick Gel Extraction Kit (Qiagen, Crawley, West Sussex, UK) and ligated into the EcoRI restriction site of the pGEM-T Easy vector (Promega, Southampton, UK) using T4 DNA ligase. The vector was transformed into the DH5 strain of Escherichia coli and bacterial plasmid preparations were obtained by the QIAprep Spin Plasmid Kit (Qiagen). The identity of each of the PCR products and hence the specificity of the PCR reaction were confirmed by DNA sequencing.
Statistical analysis
NOx values were obtained for endometrial tissue, both for control (n = 18) and study group (n = 20). The assay was performed in triplicate for each sample, a mean value was obtained and concentrations of total (NO3–) and (NO2–) were expressed as µmol/l concentrations, normalized for 0.5 mg/ml protein. Medians of NOx values from each individual phase of the menstrual cycle, were estimated. Values were assessed by a Mann–Whitney U test. RT–PCR product peaks as estimated by using the phosphorimager scanner, were performed for n = 5 and ± SE bars were obtained for each sample.
Results
NOx assays
In order to test our hypothesis that overproduction of NO is partly responsible for excessive menstrual bleeding, we measured NO indirectly by analysing the quantity of its oxidation products in uterine samples from normal and menorrhagic endometrium. NOx amounts in menorrhagic endometrial samples (n = 20) were higher than the corresponding controls (n = 18), as assessed by a Mann–Whitney U test. P < 0.01 was considered to be statistically significant (Figure 1b
).
The biopsies were randomly collected from women undergoing various phases of the menstrual cycle and the phase of each endometrial biopsy and corresponding median NOx values were recorded (Figure 1a). However, while the control and menorrhagic endometrial biopsies collected were comparable regarding stages of the menstrual cycle, we have not detected any large differences of NOx amounts depending on particular specific stages of the menstrual cycle.
Immunoblotting
The presence of iNOS has not previously been reported in the human non-pregnant myometrium. We attempted to detect iNOS in myometrial and endometrial tissue homogenates using a specific anti-iNOS antibody. A protein band of 130 kDa was detected in both endometrial and myometrial samples, corresponding to the expected size band of human iNOS, and cross-reacted with a similar-sized band in the mouse macrophage lysate positive control sample (Figure 2). Although iNOS was successfully detected in human non-pregnant endometrium and myometrium, a low signal of protein expression was obtained in both tissues, near the limit of clear detection.
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Regulation of NOS gene expression by steroids in cultured myometrial smooth muscle cells
Myocyte cultures
Explanted myometrium was treated as described earlier to give rise to primary myocyte cultures. The purity of the primary myocyte cultures was assessed by detailed comparison of the number of cells stained for
-actin to the total cell nuclei present (Figure 3a,b). Analysis of large numbers of cells indicated that ~95% of the cultured cells were identified as smooth muscle cells.
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NOS regulation by steroids in myocyte primary cultures
Quantitative RT–PCR was carried out to assess the effects of steroids upon the amounts of eNOS and iNOS RNA in myocyte primary cultures.
eNOS
Using specific eNOS primers, a PCR product of 486 bp was obtained in all reactions performed (n = 5). Nucleotide sequencing confirmed the product's identity as being human eNOS according to the known cDNA eNOS sequence. Addition of progesterone alone caused a 2.5-fold induction of eNOS RNA levels. 17ß-oestradiol combined with progesterone caused a dramatic induction of ~8-fold, whereas the `antiprogestin' RU 486 caused a slight induction, and had little or no inhibitory effect when combined with progesterone (Figures 4 and 5).
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iNOS
Using specific iNOS primers, the iNOS PCR product obtained had a size of 375 bp. Nucleotide sequencing results confirmed that the PCR product was corresponding to the expected iNOS nucleotide sequence. Steroid supplement had no effect on myocyte iNOS in vitro (Figures 4 and 6
), in contrast to the dramatic eNOS induction caused by 17ß-oestradiol and progesterone. RU 486 treatment induced iNOS mRNA levels by ~1.5-fold, while when combined with progesterone this increase was slightly inhibited.
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Discussion
This study demonstrates for the first time differences in endometrial NO amounts between women with and without menorrhagia. We also show that iNOS is present in the non-pregnant endometrium and myometrium, in addition to the previously reported uterine NOS isoforms (Telfer et al., 1997; Norman et al., 1999). In order to investigate our postulated role of steroids in regulating uterine activity and NO production, we have also examined the induction of myometrial eNOS and iNOS RNA, the main myometrial NOS isoforms, by progesterone and 17ß-oestradiol.
NOx measurements in the form of NO3– and NO2– were carried out in order to monitor indirectly the total NOS activity in the menorrhagic and control endometrium. Increased NOx concentration was linked to menorrhagia, consistent with the hypothesis that uterine NO is implicated in excessive menstrual bleeding. While our study was not designed to investigate changes in NOS activity with menstrual phase (as measured by NOx production), our data do not suggest an increase in NOS activity during menstruation; we have not detected differences on NOx amounts between phases of the menstrual cycle. According to previous work (Telfer et al., 1997), no such differences were detected in NOS isoform expression, as assessed by immunohistochemistry experiments. However, it has been suggested that endometrial Ca2+-independent NOS activity is elevated during menstruation (Tschugguel et al., 1999) and this aspect merits further investigation. In menorrhagia cases, excessive blood loss could have been a consequence of endometrial NO overproduction during the peri-menstrual phase of the cycle and endometrially-derived NO could also play a role in the local control of myometrial contractility during menstruation.
NOS immunoreactivity was initially detected in the endometrial glandular epithelium and stroma and in the myometrial blood vessels (Telfer et al., 1995), suggesting a role for NO in the paracrine control of the uterine vascular bed. eNOS and iNOS mRNA expression were later localized in isolated glands of non-pregnant endometrium and iNOS-like immunoreactivity was found in decidualized endometrial stromal cells and in first trimester pregnant tissue (Telfer et al., 1997). Progestagen in-vivo treatment of women suffering from menorrhagia showed no effect on NOS expression. Finally, eNOS and nNOS were present by immunoblotting and immunohistochemistry in pregnant and non-pregnant myometrial tissue (Norman et al., 1999). A lack of myocyte iNOS staining with occasional iNOS-expressing mast cells was previously shown (Bansal et al., 1997). The function of these cells was considered to be immunological, since iNOS was more intensely expressed by pregnant myometrial cells, indicating a role of the specific isoform in uterine quiescence.
In our attempt to determine specific NOS isoform regulation by progesterone and 17ß-oestradiol we found a marked induction of eNOS by both steroid compounds in combination, while iNOS mRNA levels were unaffected. An induction caused by progesterone was postulated to participate in the events leading to excessive menstrual bleeding. We identified this steroid induction as a property of the Ca2+-dependent isoform eNOS and not as a characteristic of the menorrhagic tissue assessed. We have not been able to test if the same steroid effect applies to cases of non-menorrhagic tissue, since all biopsies available to collect from hysterectomies originated from menorrhagia patients.
Although progesterone and 17ß-oestradiol were found to act synergistically, either progesterone or 17ß-oestradiol had an effect on myometrial eNOS induction. Our observations on oestrogens agree with the other findings (Weiner et al., 1994a) where the constitutive NOS isoforms were shown to be induced by oestrogens, and with similar published work on animal models (Figueroa et al., 1995). Oestrogens are known to induce vascular endothelial cell eNOS (Van Buren et al., 1992; Hayashi et al., 1995), promoting blood flow. In contrast, it has been demonstrated that oestrogen does not induce Ca2+-dependent NOS in human endothelial and myometrial smooth muscle cells in vitro (Tschugguel et al., 1997). The `antiprogestin' RU 486 is expected to bind with high affinity to the progesterone receptor (Catepond et al., 1997). Only a slight, partial inhibition of the progesterone effect on eNOS up-regulation, has occurred in this case. iNOS does not seem to be regulated by progesterone and 17ß-oestradiol in the case of the non-pregnant myometrium, although extensive work in pregnant rat models showed progesterone-mediated induction of iNOS, as the main isoform participating in maintenance of uterine quiescence (Buhimschi et al., 1995, 1996; Liao et al., 1996; Ali et al., 1997). We showed decrease in iNOS RNA levels by 17ß-oestradiol, in accordance with recent work suggesting inhibition of rat vascular smooth muscle iNOS by 17ß-oestradiol, via oestrogen receptor activation, indicating a novel mechanism for the protective steroid effects in cardiovascular disease (Zancan et al., 1999).
Our current findings provide evidence on the importance of the free radical gas NO in the aetiology of menorrhagia in the absence of organic disease. Endometrially-derived NO may participate in the regulation of uterine blood flow as a vasodilator and may act on the neighbouring myometrial smooth muscle cells, thus promoting uterine relaxation. The action of steroids is an additional event in myometrial relaxation, causing further production of NO by stimulating eNOS expression. Therefore, it could be suggested that administration of NO inhibitors could be an effective solution for menorrhagia patients and an alternative to surgical procedures such as endometrial ablation and hysterectomy. However, further work is necessary for better understanding of the molecular mechanisms of menorrhagia, with a view to design more specific and effective drugs towards the treatment of this common gynaecological problem.
Acknowledgments
The authors are grateful to Professor Emile-Etienne Baulieu for kindly providing RU 486. We would also like to thank the consultant gynaecologists and theatre staff at Walsgrave Hospital, NHS Trust, Coventry, West Midlands, who helped with tissue collection and all patients who participated in the study. This work was supported by the Sir Jules Thorn Charitable Trust, grant 96/02A.
Notes
3 To whom correspondence should be addressed 
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Submitted on May 28, 1999; accepted on August 10, 1999.
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