Mol. Hum. Reprod. Advance Access originally published online on February 15, 2006
Molecular Human Reproduction 2006 12(1):19-24; doi:10.1093/molehr/gah248
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Examining the spatio-temporal expression of mRNA encoding the membrane-bound progesterone receptor-alpha isoform in human cervix and myometrium during pregnancy and labour
1School of Surgical and Reproductive Sciences (Obstetrics and Gynaecology), University of Newcastle-upon-Tyne, Framlington Place, Newcastle-upon-Tyne and 2Academic Unit of Reproductive and Developmental Medicine, Sheffield Teaching Hospitals NHS Foundation Trust, Tree Root Walk, Sheffield, UK
3 To whom correspondence should be addressed at: Academic Unit of Reproductive and Developmental Medicine, Level 4, The Jessop Wing, Central Sheffield Teaching Hospitals NHS Foundation Trust, Tree Root Walk, Sheffield, South Yorkshire, S10 2SF UK. E-mail: n.r.chapman{at}sheffield.ac.uk
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Abstract |
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Human parturition is associated with a modification in the sensitivity of the myometrium to progesterone. The molecular basis for this change, however, remains unclear. It is well documented that progesterone can exert its effects through non-genomic mechanisms, including acting through membrane-bound progesterone receptors (mPRs). Recently, a novel membrane-bound PR, termed mPR
, was cloned. mPR was unlike any other PR in the databases, but it was seen to have significant homology to G-protein-coupled receptors (GPCR). In this study, we examined the spatio-temporal expression of mPR mRNA in human cervix and both lower and upper myometrial segments from non-pregnant (NP), pregnant (P) and spontaneously labouring (SL) women. We observed an incremental increase in mPR mRNA expression in NP and P samples with the peak level being observed in SL tissues. No major differences were observed between upper or lower pregnant myometrial regions. Interestingly, levels of mPR transcripts were substantially greater in labouring lower segment myometrium compared with labouring upper segment. Significantly, we failed to detect mPR message in either unripe or ripe human cervices. These data suggest that mPR protein function may play a role in regulating lower segment myometrial activity during labour. Whether it functions in the cervix, however, remains unclear. Key words: cervix/labour/myometrium/preterm/progesterone
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Introduction |
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Premature uterine contractions can result in preterm birth which, in the developed world, complicates 6–10% of all pregnancies, is responsible for over two thirds of perinatal deaths and places surviving infants at increased risk of major long-term mental and physical handicap (López-Bernal et al., 1993
; Macintosh et al., 2001; Lumley, 2003). Although this number may seem small, it has a disproportionate impact on the health care budgets of many countries (Gill, 2001); the United Kingdom health service spends between £42 and 74 million per annum on neonatal intensive care for babies weighing less than 1500 g (Hall et al., 1997) although this is small when compared with the human cost involved. Moreover, current medical therapies have limited use and are associated with serious side effects for both the infant and the mother (Higby et al., 1993). Because the intrauterine health of a baby is increasingly seen as a major predictor of adult morbidity, the ability to deliver a healthy child, at term, is critical considering those socioeconomic costs associated with such adult diseases (Gill, 2001).
The ability of progesterone to block myometrial activity in response to estrogen has been known for 40 years (Csapo and Pinto-Dantas, 1965). In many mammalian species, progesterone withdrawal is a prerequisite for labour, and the mechanism by which the uterus becomes insensitive to progesterone at term is complex and species specific. In some mammals (mice, rats, rabbits, goats and pigs), labour is associated with a reduction in the level of circulating progesterone associated with the regression of the progesterone-secreting corpus luteum (Astle et al., 2003). In sheep, the mechanism of circulatory progesterone reduction involves the up-regulation of the placental enzyme 17--hydroxylase which catalyses the conversion of progesterone into estrogen thereby facilitating the reduction in circulating progesterone levels (Anderson et al., 1975). In both humans and non-human primates, however, the method of progesterone withdrawal is unclear—no decline in plasma progesterone being observed at term (Broditsky et al., 1978; Walsh et al., 1984). There is also conflicting data describing the expression of the nuclear progesterone receptors (PRs) in reproductive tissues with some groups observing a decline in receptor at term (Khan-Dawood and Dawood, 1984; Padayachi et al., 1990; How et al., 1995; Rezapour et al., 1997), whereas others report an increase (Bernard et al., 1988; Winkler et al., 2002). Furthermore, various data suggest that changes in the ratio of different nuclear PR isoforms (Pieber et al., 2001a,b; Mesiano et al., 2002) association of the nuclear PR with Nuclear Factor kappaB (NF-
B; Allport et al., 2001), competition between cortisol and progesterone for the nuclear PR (Karalis et al., 1996) or a reduction in the levels of steroid receptor co-activators at term (Condon et al., 2003) may underpin the functional progesterone withdrawal observed in humans. To date, however, none of these hypotheses have been unequivocally accepted.
The uncertainty regarding the physiological function of human progesterone withdrawal translates into the questionable clinical effectiveness of progesterone as an agent to prevent preterm birth. For example, a relatively recent clinical trial administering 17--hydroxyprogesterone caproate to women deemed to be at high risk of premature labour was associated with a reduced incidence of preterm delivery (Meis et al., 2003). This treatment regime, however, was only effective in women who have had previous spontaneous premature deliveries not those in other high-risk groups (Meis, 2005).
Progesterone generally exerts its effects through induction of transcriptional events, mediated by cytosolic steroid receptors belonging to the superfamily of nuclear transactivators (Weigel, 1996). Because this has been reviewed extensively elsewhere (Bramley, 2003; Leonhardt et al., 2003), it will not be addressed here.
It is increasingly apparent, however, that progesterone and other steroids including estrogen, aldosterone and vitamin D can exert their effects through non-traditional means which are seen as rapid effects on target cells induced within 10 min (Leonhardt et al., 2003; Lösel and Wehling, 2003; Lösel et al., 2003). Such effects, including changes in intracellular Ca2+ or cAMP, (Maller and Krebs, 1977; Sanchez-Bueno et al., 1991) are not compatible with the classical scheme of steroid action detailed above and in a previous study (Weigel, 1996) and have been described in many cell types (Blackmore and Lattanzio, 1991; Blackmore et al., 1991; Gerdes et al., 1998; Luconi et al., 1998; Falkenstein et al., 2000; Bagowski et al., 2001; Boonyaratanakornkit et al., 2001; Lösel et al., 2004; Shah et al., 2005; Skildum et al., 2005).
Significantly, Perusquía and Jasso-Kamel (2001) demonstrated that various progestins could elicit rapid and reversible relaxatory effects on isolated strips of human myometrium obtained at term. These effects could not be inhibited by the nuclear PR antagonist, RU486, suggesting the involvement of a myometrial, non-genomic membrane PR (Perusquía and Jasso-Kamel, 2001). Importantly, a recent study demonstrated that a dose of 10–5 M natural progesterone could significantly reduce the amplitude of contraction in isolated, oxytocin-stimulated human myometrial strips (Chanrachakul et al., 2005). Moreover, a dose of 10–8 M natural progesterone was seen to augment the ritodrine-induced myometrial relaxation suggesting that progesterone can exert its effects in myometrium through a non-genomic mechanism, possibly involving G-protein-coupled receptors (GPCR) (Chanrachakul et al., 2005).
Several membrane-bound PRs have been reported (Blackmore and Lattanzio, 1991; Lösel and Wehling, 2003; Lösel et al., 2004; Shah et al., 2005). Each is predicted to possess a single membrane spanning domain and to be linked to an ion channel of some description (Lösel and Wehling, 2003). For example, in human sperm exposed to progesterone rapid influxes of Ca2+ and effluxes of Cl– ions are observed facilitating the induction of acrosomal exocytosis (Wistrom and Meizel, 1993; Aitken et al., 1996; Sabeur et al., 1996). More recently, however, a novel membrane-bound PR was cloned from oocytes of the spotted sea trout, Cynoscion nebulosus (Zhu et al., 2003a) which was subsequently shown to have three human homologues termed membrane-bound progesterone receptor-alpha (mPR), mPRß and mPR
(Zhu et al., 2003b). Moreover, these novel mPRs share very little homology with those single-span PRs described above. For example, mPR, mPRß and mPR all possess seven membrane-spanning domains as opposed to one and share some biochemical characteristics with the GPCR rather than ion channels. Of these proteins, only mPR was associated with reproductive tissues—namely placenta; cervix and myometrium were not considered. Consequently, the aim of this study was to define the spatio-temporal expression pattern of mPR mRNA transcripts in human cervix and both lower and upper segment myometrium.
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Materials and methods |
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Selection of patients and tissue collection
All women were recruited from the Department of Obstetrics and Gynaecology at the Royal Victoria Infirmary, Newcastle-upon-Tyne. This study received approval from the Newcastle and North Tyneside Health Authority Ethics Committee, and all patients gave written, informed consent.
Non-pregnant myometrium
Myometria were obtained from non-pregnant (NP) premenopausal women (n = 6, aged 32–46) undergoing hysterectomy for benign gynaecological conditions. The samples were taken immediately following the removal of the uterus. The lower segment only appears late in pregnancy and is not present in the NP uterus. Therefore, to facilitate topographical comparisons in this study, NP uteri were sampled from the lower corpus well clear of the cervix with myometrium being taken from the middle of the uterine wall, allowing generous clearance margins from the serosal and endometrial surfaces. Samples obtained in both the follicular and luteal phase of the cycle were used.
Term pregnant myometrium
Upper and lower uterine segment myometrial samples were obtained from healthy women undergoing elective caesarean section at term (n = 14, age 16–43, gestation 37–40 weeks). The indications for section were breech presentation or previous caesarean section. Excluded from this group were women whose cervices had dilated beyond 2 cm or who were experiencing regular painful uterine contractions. Patients who had had prostaglandin (PG) gel administered or whose amniotic membranes were not intact were also excluded. The samples were obtained immediately following the delivery of the placenta and membranes before the closure of the uterine cavity. Samples from the upper uterine segment were taken under direct vision using Wolf biopsy forceps introduced into the uterine cavity through the incision (Robson et al., 2002). The forceps were pushed through the decidual layer and into the myometrium. Eight separate myometrial biopsies were taken from individual patients, each from a non-placental bed site (as determined by manual palpation before the delivery of the placenta). Samples from the lower uterine segment were taken from the upper lip of the incision through the lower uterine segment using toothed biopsy forceps to grasp the myometrium from between its serosal and decidual layers then curved scissors to sample it.
Term labouring myometrium
Upper and lower segment myometrial samples were obtained from women admitted in spontaneous labour (SL) (defined as the onset of painful regular uterine activity resulting in the progressive and serial dilatation of the cervix beyond 3 cm) undergoing emergency caesarean section at term (n = 14, age 16–41, gestation 37–42 weeks, median 40). Indications for section were failure to progress and fetal distress. Women who had their labour induced or augmented before reaching 3 cm were excluded from the study. Upper and lower segment biopsies were obtained in a similar manner to those from women undergoing elective caesarean section. All myometrial samples were snap frozen at the time of collection using liquid nitrogen cooled iso-pentane and then stored at –70°C.
Unripe and ripe cervix
Punch biopsies of the pregnant cervix were obtained from patients undergoing elective caesarean section at term (n = 6, age 16–43, gestation 37–40 weeks) as detailed in Sakamoto et al. (2004). No cervical dilatation was evident at the time of biopsy. Cervices were graded by an experienced obstetrician for ripeness and given an appropriate Bishop score (
3 for unripe; 
8 for ripe).
Preparation of total RNA
Total RNA was prepared from human cervical and myometrial biopsies using TRI-reagent (a monophasic solution of phenol and guanidine thiocyanate; catalogue number T9424; Sigma-Aldrich, Saint Louis, Missouri USA) according to the manufacturer’s protocol. The resulting pellet was resuspended in 100 µl of RNAse-free water. This total RNA was then applied to an RNeasy column (Qiagen, GmbH, Hilden, Germany) to facilitate DNAseI treatment thereby removing any residual genomic DNA. Again the manufacturer’s guidelines were followed. Total RNA was then eluted in 35 µl of RNAse-free water. Expression of mPR message was then determined using RT–PCR analyses with the following primers generating an internal fragment from nucleotide 204–783: mPR sense 5'-CACAACGAGGCCGTGAATGTC-3' and antisense 5'-GCTCGGGCATGAAGGTAGAG-3'. To ensure equal loading of samples, a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was amplified using the following primers: GAPDH sense 5'-CTGCCGTCTAGAAAAACC-3' and antisense 5'-CCACCTTCGTTGT CATACC-3'. To amplify nuclear PR-B message, the following primers, generating an internal fragment from nucleotide 132–332, were synthesized: PR-B sense 5'-CCTGAAGTTTCGGCCATACCT-3' and antisense 5'-AGCAGTCCGCTGTCCTTTTCT-3'. This fragment is unique to PR-B as it resides within the extreme N-terminal of the PR message and ensured only PR-B was recovered.
All RT–PCRs utilized the rapid access RT–PCR kit from Promega (Southampton, UK). All RT–PCR experiments were performed in the linear range, and the target sequenced to verify it was mPR. Reaction conditions were as follows: 1 cycle of reverse transcription at 48°C for 45 min followed by 30 cycles at 95°C for 1 min, 55°C for 1 min and 72°C for 1 min. This was then followed by 1 cycle of a final extension at 72°C for 10 min. Products were visualized using 2% Tris-acetate-EDTA agarose gel electrophoresis (TAE-AGE) with cDNAs of 
579 bp (mPR), 200 bp (PR-B) and 220 bp (GAPDH) being obtained.
Statistical analyses
Data were compared using one-way analysis of variance (ANOVA) followed by a Bonferroni multiple comparisons post test; P < 0.05 was considered statistically significant. All experiments were performed three times, and results are expressed as the mean ± SEM.
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Results |
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Temporal expression of mPR
mRNA in lower segment myometrial biopsies from NP, P and SL womenUsing semiquantitative RT–PCR, we examined the expression profile of mPR
mRNA in myometrial biopsy samples from lower uterine segments from P and SL women and myometrium from the lower corpus, well clear of the cervix, from NP women. Interestingly, mRNA encoding this receptor was seen to increase sequentially in NP and P tissues reaching a maximum in SL samples (Figure 1A). All samples were equally loaded as judged by the uniform expression of GAPDH (Figure 1B). No signal was detected when reactions were performed without reverse transcriptase suggesting that all samples were free from genomic DNA contamination (not shown).
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Spatial expression of mPR transcripts in upper (corpus) and lower uterine segment myometrial biopsies from P and SL women
To determine whether topographical distributions of mPR transcripts occurred in different regions of the uterus during pregnancy and labour, RT–PCR was performed on total RNA isolated from myometrial tissue sampled from the upper (corpus) and lower uterine segments from P and SL women. No differences in expression of mPR mRNA were seen between pregnant upper and lower samples (Figure 2A). Interestingly, however, an increase in mPR mRNA was observed in SL lower segment compared with SL upper segment (Figure 2A). No statistical difference in GAPDH expression in all upper and lower segment myometrial samples was observed (Figure 2B).
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mPR mRNA is undetectable in both unripe and ripe cervix
Progesterone exerts its effects on both myometrium and cervix with the nuclear PR being observed in both tissues (Stjernholm-Vladic et al., 2004). To assess whether mPR message was present in either unripe or ripe human cervix, RT–PCR analysis was performed on total RNA isolated from cervical biopsies from patients with a low (unripe; 3) or high Bishop score (ripe; 8). Matched lower uterine segments were also analysed in this series of experiments to serve as positive controls although it should be stressed that no direct quantitative comparison between cervix and myometrium can be made because of the differences in tissue composition. Significantly, mRNA encoding mPR could not be detected in either matched unripe or ripe cervices (Figure 3A and C) suggesting mPR expression and activity may only be of functional importance within the myometrium. Again, no statistically significant difference in expression of GAPDH was observed between samples (Figure 3C and D).
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To ensure that the lack of mPR message in the cervical samples was not because of general, non-specific destruction of RNA, we analysed expression of mRNA encoding the nuclear PR, PR-B, by RT–PCR. Because this was a control experiment, we decided to focus on PR-B as this has been previously shown to be present in both cervix (Stjernholm-Vladic et al., 2004) and myometrium (Mesiano et al., 2002). PR-B was seen to be expressed at equivalent levels in both cervical and lower segment myometrial samples (Figure 4A and C) suggesting the inability to detect mPR mRNA in the cervix is not because of a global down-regulation of gene expression in this tissue. Again, GAPDH expression was equal between samples (Figure 4B and D).
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Discussion |
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Spatio-temporal expression of mPR
mRNAWe have examined the expression of mRNA encoding a novel membrane-bound PR, mPR
, in human cervix and both lower and upper myometrial regions. Our findings illustrate that mRNA for this receptor is present throughout the myometrium but undetectable in either unripe or ripe cervices. Significantly, an incremental increase in mPR transcript expression was detected between NP and P samples with a further increase being documented in labouring lower segment myometrium. We should stress at this juncture, however, that a given number of mPR transcripts may not accurately reflect the level of functional mPR protein. Interestingly, our observation is in direct contrast to that recently reported by Fernandes et al. (2005) who described a 50% reduction in mPR mRNA in SL lower segment human myometrium. The reason for this disparity is not clear at present.
There appeared to be a topographical distribution of myometrial mPR message within different regions of the uterus, because expression levels of this transcript were significantly higher in lower, compared with upper myometrial biopsies from SL women. It is well established that the upper and lower regions of the uterus govern contractility and dilatation, respectively, and there is an increasing body of data describing differential expression of proteins within these distinct uterine regions. Examples would include COX-1 and -2 (Sparey et al., 1999), PGE2 receptors (Astle et al., 2005), NF-B (Chapman et al., 2004), oxytocin receptors (Fuchs et al., 1984) and corticotrophin-releasing hormone receptors (Stevens et al., 1998).
Detection of mPR message would suggest the receptor is expressed although, as stated above, one must appreciate that it is virtually impossible to correlate most mPR transcripts with the amount of functional mPR receptor protein because little is known about the stability of either the mPR mRNA or the mPR protein associated with the membrane. Moreover, if such studies were to be attempted at present, many difficulties would arise. For example, there are no commercially available antisera with which to perform a series of western analyses to quantitate mPR at the protein level or immunohistochemical studies to directly visualize this receptor in the myometrium. The only commercial source of an anti-mPR serum is raised against mPR not mPR, and the specificity of this material has not been rigorously verified. Furthermore, a second, non-commercial source of an anti-mPR serum (Fernandes et al., 2005) failed to detect mPR in western analysis, and the epitope used shared significant homology to both mPRß and mPR, therefore raising questions about the specificity of this reagent.
Although membrane-impermeant progesterone [fluorescein isothiocyanate–bovine serum albumin-progesterone (FITC-BSA-progesterone) conjugates] can be used to directly visualize progesterone binding to the surface membranes (Blackmore and Lattanzio, 1991; Luconi et al., 1998; Shah et al., 2005), there have been suggestions that such membrane-impermeant progesterone analogues can still penetrate the plasma membrane to some degree (Bernard et al., 1957; Moats and Ramirez, 2000; Nishimura and Nakano, 2000) and that some cell types may possess few different membrane-bound PRs (Gerdes et al., 1998; Lösel et al., 2004). Furthermore, using peroxidase-conjugated progesterone in ligand blot analyses would merely identify all proteins capable of binding progesterone—it would fail, unequivocally, to determine whether the progesterone-binding protein identified was mPR. This approach would need to be coupled to an in-depth proteomic analysis using MALDI-TOF (Matrix Assisted Laser Desorption/Ionization-Time-Of-Flight) mass spectral analysis of tryptic digests of those progesterone-binding proteins.
Functional consequence of mPR protein expression
The biochemical function of the mPR receptor protein in human myometrium could not be addressed here and consequently remains elusive. In their original study describing the isolation of mPR, Zhu et al. (2003b) highlighted that it may serve as a GPCR. In keeping with this postulate, those authors also illustrated that progesterone signalling through this receptor was seen to dramatically reduce intracellular cAMP formation in the breast cancer cell line MDA-MB-231. This effect could be inhibited by pertussis toxin suggesting the involvement of the Gi GTP-binding protein (Zhu et al., 2003b). Signalling through Gi would reduce myometrial cAMP levels, and such reductions in cAMP have been associated with the termination of myometrial quiescence (Europe-Finner et al., 1993, 1994). The MDA-MB-231 line, however, is an estrogen-receptor negative cell known to have constitutively active NF-B (Nakshatri et al., 1997). Because both NF-B and estrogen are connected with uterine progesterone signalling (Kalkhoven et al., 1996; Mesiano et al., 2002), it would seem prudent to exercise caution when extrapolating the observations of Zhu et al. (2003b) to primary human myometrium. Whether mPR couples to Gi or other GTP-binding proteins, including Gs, in myometrium is unclear and definitely warrants further investigation.
Interestingly, an elevated expression of mPR protein in labouring lower segment myometrium would provide more progesterone binding sites within this region. Whether such sites actively compete with the nuclear PR isoforms for progesterone, thereby switching from one signalling cascade to another, is presently undefined. For example, increased stimulation of mPR by progesterone may serve as the initiating event leading to the reduction in steroid receptor co-activator levels that are observed in the labouring mouse uterus (Condon et al., 2003). Alternatively, signalling through mPR receptor protein may serve to modulate nuclear-receptor function, by inducing nuclear-receptor phosphorylation (Zhang et al., 1997; Clemm et al., 2000) or other post-translational modifications known to modulate transcription factors including receptor protein acetylation and methylation (Bannister and Kouzarides, 2005). This would permit the fine tuning of nuclear PR-regulated gene expression, which must occur before parturition, as opposed to there being a crude binary switch. Such notions, however, still leave unanswered the question of how mPR mRNA levels are increased, and our future work aims to answer this question.
In conclusion, our study illustrates that mRNA encoding a novel membrane-bound PR, mPR, is present in human myometrium but undetectable in cervix and that expression of mPR message increases incrementally with the peak being observed in lower labouring myometrial segment tissues.
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Acknowledgements |
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The authors are grateful to Drs Andrew Loughney and Malcolm MacDougall along with patients and midwives at the RVI for providing/obtaining myometrial biopsies. This work was funded by the University of Newcastle-upon-Tyne (N.R.C.). G.N.E.F. is funded by grants from Action Medical Research and the Wellcome Trust.
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Notes |
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* The authors contributed equally to this work.
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References |
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Submitted on November 16, 2005; accepted on November 21, 2005.
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