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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2007-0077
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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 5 1927-1933
Copyright © 2007 by The Endocrine Society

Nuclear Progesterone Receptors in the Human Pregnancy Myometrium: Evidence that Parturition Involves Functional Progesterone Withdrawal Mediated by Increased Expression of Progesterone Receptor-A

Amy A. Merlino, Toni N. Welsh, Huiqing Tan, Li Juan Yi, Vernon Cannon, Brian M. Mercer and Sam Mesiano

Department of Reproductive Biology, Case Western Reserve University, and Department of Obstetrics and Gynecology (T.N.W., H.T., L.J.Y., S.M., V.C.), University Hospitals, Case Medical Center, Cleveland, Ohio 44106; and Department of Obstetrics and Gynecology (A.A.M., B.M.M.), MetroHealth Medical Center, Cleveland, Ohio 44109

Address all correspondence and requests for reprints to: Sam Mesiano, Ph.D., Department of Reproductive Biology, Case Western Reserve University, 11100 Euclid Avenue, Cleveland, Ohio 44106-5034. E-mail: sam.mesiano{at}case.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: We examined whether human parturition involves functional progesterone withdrawal mediated by changes in myometrial expression of progesterone receptors (PRs)-A and -B.

Objective: Our objectives were to: 1) measure PR-A and PR-B protein levels in human pregnancy myometrium and determine whether the PR-A to PR-B ratio changes with advancing gestation and labor onset; and 2) determine how changes in the PR-A to PR-B ratio affect myometrial cell progesterone responsiveness.

Design: PR protein levels and cellular localization were measured by Western blotting and immunohistochemistry, respectively, in lower uterine segment uterine wall tissue from preterm (<37 wk; not laboring; n = 5) and term (37–40 wk; not in labor: n = 6; in labor: n = 5) cesarean delivery. The capacity for PR-A and PR-B, alone and in combination, to mediate genomic progesterone responsiveness measured by the activity of a progesterone-responsive reporter plasmid was examined by artificially modulating their levels in the PHM1–31 myometrial cell line.

Results: PR-A and PR-B immunostaining was detected only in the nucleus of myometrial cells. The PR-A to PR-B protein ratio was 0.49 ± 0.082 (mean ± SEM) in preterm tissue; increased to 1.03 ± 0.071 (P < 0.001) in nonlaboring term tissue; and increased further to 2.65 ± 0.344 (P < 0.001) in laboring term tissue. Only PR-B mediated progesterone-induced transcriptional activity. PR-A had no effect alone but markedly decreased PR-B-mediated progesterone responsiveness.

Conclusions: Functional progesterone withdrawal in human parturition may be mediated by an increase in the myometrial PR-A to PR-B ratio due to increased PR-A expression.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
PROGESTERONE MAINTAINS PREGNANCY mainly by promoting myometrial relaxation. Treatments that interfere with progesterone action or synthesis induce labor, and it is generally considered that natural labor is initiated by progesterone withdrawal. In most species, progesterone levels fall precipitously before the onset of labor, and this systemic progesterone withdrawal is thought to be a key parturition-triggering event. Human parturition, however, occurs without a systemic progesterone withdrawal; instead maternal, fetal, and amniotic fluid progesterone levels remain elevated during labor and delivery (1, 2, 3). To explain this conundrum, it is hypothesized that human parturition involves a functional progesterone withdrawal mediated by decreased myometrial progesterone responsiveness.

Progesterone responsiveness is controlled primarily by the extent of nuclear progesterone receptor (nPR) expression. The human nPR exists as two major types: the full-length progesterone receptor (PR)-B and the N-terminal truncated PR-A (4, 5, 6). A further truncated nPR, PR-C, has also been reported; however, its role is unclear (7, 8, 9). In vitro studies have shown that PR-B is the principal mediator of progesterone actions (10, 11, 12), whereas PR-A represses the transcriptional activity of PR-B (10). In most cells, the extent to which PR-A represses PR-B-mediated transcriptional activity is directly related to its abundance relative to PR-B (i.e. the PR-A to PR-B ratio). Based on this model, we hypothesize that during most of human pregnancy progesterone promotes myometrial relaxation through its interaction with PR-B and that functional progesterone withdrawal at parturition is mediated by increased myometrial expression of PR-A.

We previously tested this hypothesis using quantitative RT-PCR (qRT-PCR), and our data suggested that in term myometrium the PR-A to PR-B mRNA ratio increases with labor onset primarily due to increased expression of PR-A (13). However, those conclusions were equivocal because our assay could not discriminate between transcripts encoding PR-A or PR-C. Others also examined the PR-A/PR-B hypothesis by measuring nPR protein levels in the human pregnancy myometrium. Pieber et al. (14) reported that PR-A increases markedly in association with the onset of labor, whereas PR-B was high and not affected by labor status. Condon et al. (15) also reported robust levels of PR-B in the pregnancy myometrium; however, they could not detect PR-A. Instead, they detected a smaller immunoreactive protein, which they attributed to be PR-C. The putative PR-C was detected only in the cytoplasmic fraction of fundal myometrium, and its abundance increased markedly with the onset of labor. They proposed that functional progesterone withdrawal in human parturition is mediated by an increase in the myometrial PR-C to PR-B ratio, rather than the PR-A to PR-B ratio and that this occurs predominantly in the uterine fundus. However, the specificities of the respective nPR antibodies used for Western blotting in studies by Pieber et al. (14) and Condon et al. (15) were not confirmed, and the identities of the nPR protein bands were not verified. Moreover, the robust levels of PR-B detected in both studies were inconsistent with the very low PR-B mRNA levels measured by qRT-PCR, and the lack of PR-A or PR-C reported by Condon et al. in lower section myometrium is inconsistent with our detection of mRNAs in this tissue that could encode these proteins. Thus, the types of nPRs expressed in the human pregnancy myometrium and how their levels change in association with the onset of labor is uncertain. To address this issue, we used a highly sensitive and specific nPR Western blot assay to determine whether PR-A and PR-B are present in term human pregnancy myometrium and, if so, whether their levels change with advancing gestation and the onset of labor. We also determined the cellular localization of PR-A and PR-B in myometrial specimens and conducted a series of in vitro studies to assess how PR-A and PR-B alone and in combination mediate genomic progesterone responsiveness in a myometrial cell line.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Myometrial tissue specimens

Myometrial tissue (approximately 0.5 cm3) was obtained from consenting women undergoing cesarean delivery at MacDonald Women’s Hospital, University Hospitals, Case Medical Center (institutional review board no. 11-04-06) and MetroHealth Medical Center (institutional review board no. IRB05–00287). Tissue was excised from the upper margin of the transverse incision made in the lower uterine segment. Three clinical groups were examined: 1) women at term (37–40 wk) and not in labor (TNIL; n = 6) having an elective cesarean delivery; 2) women at term and in labor (TIL; n = 5) exhibiting regular and forceful contractions coupled with cervical dilation greater than 4 cm and/or documented cervical change, who required a cesarean delivery for reasons independent of uterine contractions (e.g. breech presentations, previous cesarean delivery, fetal distress); and 3) women having a cesarean delivery preterm (<37 wk) who were not in labor and who required a cesarean delivery for conditions that did not affect myometrial contractility (e.g. nonreassuring fetal status) (PTNIL; n = 5; range 24–33 wk). Tissue specimens were washed in ice-cold PBS, dissected from decidua and connective tissue, and divided into two pieces: one was snap frozen in liquid nitrogen and stored at –80 C, the other was placed into fixative (4% paraformaldehyde in PBS) for 24 h at 4 C and then paraffin embedded.

Cell culture

The PHM1–31 immortalized human myometrial cell line was provided by Dr. Barbara Sanborn (Colorado State University) (16). We have found that PHM1–31 cells express very low levels of PR-A and PR-B and do not respond genomically to progesterone. The T-47D breast cancer cell line (American Type Culture Collection, Manassas, VA), which expresses very high levels of PR-A and PR-B, was also used. Cell cultures were maintained at 37 C in 95% air-5% CO2 in phenol red-free DMEM containing 5% charcoal-stripped fetal calf serum, 0.1 mg/ml geneticin, 2 mM L-glutamine, and antibiotics (penicillin/streptomycin). Medium was refreshed every 48 h.

Nucleofection of expression plasmids and small interfering RNAs (siRNAs)

Cytomegalovirus promoter-driven expression plasmids for PR-A and PR-B (17) and a progesterone-responsive luciferase reporter plasmid (PRE-Luc) (18) were provided by Dr. Zafar Nawaz (University of Miami, Miami, FL). Nuclear PR siRNA, which induces RNA interference on all nPR transcripts, was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Expression plasmids and siRNAs were introduced into cell lines by nucleofection (Amaxa Inc., Gaithersburg, MD). Briefly, cells were harvested by trypsinization, centrifuged, and resuspended in smooth muscle nucleofection solution (Amaxa) to a concentration of 2 x 106 cells per 100 µl. The mixture was then transferred into an electroporation cuvette, placed in the Nucleofector device (Amaxa), and subjected to program A33. We previously determined that this program produces a transfection efficiency of 30–40% in PHM1–31 and T-47D cells. Cells were then replated. Cells transfected with PRE-Luc (and pRenilla-Luc used to normalize for transfection efficiency) were allowed to stabilize for 16 h and then exposed to 100 nM progesterone (Sigma, St. Louis, MO) or vehicle for 8 h. Cells were then processed to measure relative luciferase activity using the dual-luciferase reporter assay system (Promega Inc., Madison, WI).

Western blot analysis

Myometrial tissue specimens were homogenized in CelLytic MT extraction buffer (Sigma), supplemented with protease inhibitors and phosphatase inhibitors (final concentrations: 0.5 mmol/liter phenylmethylsulfonyl fluoride, 86 µmol/liter leupeptin, 77 µg/ml aprotinin, 1.4 µmol/liter pepstatin A, and 100 µg/ml bacitracin) on ice. Homogenates were centrifuged at 1000 x g for 10 min at 4 C and supernatants collected and assayed for protein content using the Bradford protein assay (Pierce, Rockford, IL). Cell lysates (50–100 µg) were diluted in gel loading buffer [40% glycerol, 1 M Tris-HCl, 2.5% ß-mercaptoethanol, 8% sodium dodecyl sulfate, and 0.01% bromophenol blue], heated for 5 min at 100 C, and subjected to denaturing (in 10% SDS-PAGE) using the NuPAGE Bis-Tris system (Invitrogen, Carlsbad, CA). Separated proteins were then transferred to polyvinyl difluoride membrane (Bio-Rad Laboratories, Hercules, CA). Blots were blocked with 5% nonfat milk in 20 mM Tris, 500 mM NaCl (pH 7.5) containing 0.05% Tween 20 (TTBS) for 1 h at room temperature and then incubated with primary antibodies [anti-nPR PgR1294, 1:625 (Dako North America, Inc., Carpinteria, CA); anti-nPR sc-538, 1:1000 (Santa Cruz); anti-h-CAL, 1:2000 (Neomarkers; Lab Vision Corp., Fremont, CA); antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH), 1:50000 (Santa Cruz)] overnight at 4 C. The following day, blots were washed three times with TTBS and incubated at room temperature with an horseradish peroxidase-conjugated antimouse secondary antibody (Cell Signaling, Boston, MA). The blots were then treated with chemiluminescent reagents (Amersham Life Sciences, Piscataway, NJ) and exposed to x-ray film (Hyperfilm ECL; Amersham Life Sciences). Films were scanned and intensity of bands quantified using digital densitometry (1D Image software; Kodak, Rochester, NY).

Immunohistochemistry (IHC)

Paraffin sections were deparaffinized in xylene and rehydrated in graded ethanols. Sections were heated at 95 C in citrate buffer [10 mM citric acid, 0.05% Tween 20 (pH 6.0)] for 20 min, cooled to room temperature, and rinsed in TTBS. Sections were pretreated for 5 min in 3% H2O2, washed, and then incubated with anti-nPR PgR1294 (Dako), anti-h-CAL (Neomarkers), or nonimmune mouse IgG (Santa Cruz) (each 1:250 diluted in 1% BSA) at room temperature for 1 h. Sections were washed and then incubated with biotinylated antimouse IgG (Dako) for 20 min at room temperature, washed again, and incubated with streptavidin-horseradish peroxidase for 20 min at room temperature and then incubated with 3,5 diaminobenzidine tetrahydrochloride, rinsed in water, dehydrated in graded alcohols, cleared in xylene, and mounted with coverslips. Some sections were stained with hematoxylin or eosin before ethanol dehydration and mounting.

Statistical analyses

nPR protein levels in tissues from the three clinical groups were compared by one-way ANOVA using the Kruskal-Wallis ranked test. Differences between experimental groups were assessed by Student’s t and the Mann-Whitney tests. Differences were considered statistically significant when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Specificity of nPR Western blotting

To determine whether proteins detected by PgR1294 in myometrial cells are nPRs, we artificially overexpressed PR-A and PR-B in PHM1–31 cells and subjected the transfected cells to PgR1294-Western blotting. In untransfected cells or cells transfected with an empty expression plasmid, nPR levels were very low and barely detectable. As expected, in PHM1–31 cells transfected with PR-A and PR-B expression plasmids, PgR1294 reacted specifically with proteins of predictable molecular mass for PR-A (80–90 kDa) and PR-B (100–110 kDa). These proteins corresponded with the typical PR-A and PR-B bands detected in T-47D cells (Fig. 1AGo).


Figure 1
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  		FIG. 1. Specificity of the PgR1294 nPR antibody. A, Western blot analysis of lysates (50 µg) from PHM1–31 cells engineered to overexpress PR-A or PR-B. Empty vector was used as a transfection control. T-47D lysate (0.3 µg) was included as a positive control for PR-A and PR-B. The blot was probed simultaneously with PgR1294 (1:625) and anti-GAPDH (1:50,000). B, RNA interference of nPR transcripts in T-47D cells results in a decrease in the intensity of bands corresponding to the PR-A and PR-B proteins detected by PgR1294. C, Comparison of the sc-538 (1:1000) and PgR1294 (1:1000) nPR antibodies for the detection of PR-A and PR-B by Western blotting of term myometrial lysate (each 50 µg).

 
To further test the specificity of the PgR1294 antibody, we measured nPRs by PgR1294-Western blotting in T-47D cells subjected to RNA interference for nPRs. The proteins corresponding to PR-A and PR-B decreased by around 50% in cells transfected with nPR siRNA (Fig. 1BGo).

We also compared the PgR1294 antibody with another commercially available and commonly used rabbit polyclonal nPR antibody, sc-538 (Santa Cruz). We found that PgR1294 detects PR-A and PR-B in term myometrium lysate, whereas sc-538 does not detect PR-A and PR-B but instead interacts with two proteins with apparent molecular mass of 100 and 50–60 kDa (Fig. 1CGo).

Cellular localization of nPR in uterine wall specimens

We used the PgR1294 antibody to determine the cellular localization of PR-A and PR-B in myometrial specimens (Fig. 2Go). To identify smooth muscle cells, serial sections were subjected to IHC using an antibody to high molecular weight caldesmon (h-CAL), a thin filament-associated protein expressed only by smooth muscle cells (19, 20). PgR1294 immunoreactivity was detected predominantly in the nucleus of h-CAL-positive myometrial cells. h-CAL IHC analyses revealed that despite our efforts to collect pure myometrium, the amount of nonmyometrial tissue in each specimen varied considerably.


Figure 2
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  		FIG. 2. Localization of PR-A and PR-B (by PgR1294) and h-CAL in lower segment uterine tissue specimens. Left and middle columns are serial sections subjected to IHC without (left) and with (middle) eosin counter staining. Right column shows sections from another specimen subjected to IHC for h-CAL and PgR1294 and counterstained with hematoxylin. The magnified inset shows the exclusive localization of PR-A and PR-B in the nucleus of myometrial cells. Nonimmune mouse IgG control is shown in the lower row. Myo, Myometrial bundles; vsm, vascular smooth muscle. Bar, 100 µm.

 
nPR levels in the pregnancy myometrium

Consistent with our IHC findings, our Western blotting showed that lower segment myometrial tissues contain PR-A and PR-B proteins (Fig. 3AGo). Because PR-A and PR-B are expressed only in h-CAL-positive cells and abundance of h-CAL in the human pregnancy uterus is not affected by stage of gestation or labor status (21), PR-A and PR-B protein levels were normalized to h-CAL (normalization to GAPDH was problematic due to presence of varying amounts of nonmyometrial tissue). Relative levels of PR-B remained constant in each experimental group, whereas levels of PR-A were higher in TNIL vs. PTNIL (P < 0.05) tissues and in TIL vs. TNIL or PTNIL tissues (Fig. 3BGo; both P < 0.05). The PR-A to PR-B ratio favored PR-B in PTNIL samples by approximately a 2:1 ratio (PR-A to PR-B ratio: 0.49 ± 0.082; mean ± SEM). At term, levels of PR-A, but not PR-B, increased, compared with PTNIL levels, and consequently, the PR-A to PR-B ratio increased significantly to 1.03 ± 0.071 (P < 0.001). The onset of term labor was associated with a further increase in PR-A levels with no change in PR-B levels, and the PR-A to PR-B protein ratio increased to 2.65 ± 0.344 (P < 0.001) (Fig. 3CGo). In two specimens derived from women in advanced active labor, two immunoreactive proteins were detected at around 70 and 80 kDa. The identity of these proteins is uncertain.


Figure 3
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  		FIG. 3. Analysis of PR-A and PR-B proteins in lower segment PTNIL, TNIL, and TIL myometrium specimens. A, Western blot analysis of myometrial lysates (100 µg per lane; each lane represents a different specimen) probed with anti-h-CAL (upper panel) and PgR1294 (lower panel). A T-47D lysate (1.5 µg) lane is included as an nPR-positive control. B, Abundance (assessed by digital densitometry of Western blot shown in A) of PR-A and PR-B relative to h-CAL. C, PR-A to PR-B densitometric ratio. Each bar, mean ± SEM (*, P < 0.05; **, P < 0.001).

 
nPR function in myometrial cells

Progesterone elicited a marked increase in PRE-Luc activity only in cells overexpressing PR-B; overexpression of PR-A failed to confer genomic progesterone responsiveness (Fig. 4Go). As expected, PR-A decreased the transcriptional activity of PR-B in PHM1–31 cells. The PR-A to PR-B ratio achieved in the cotransfected cells varied between 1.5 and 3.0.


Figure 4
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  		FIG. 4. Effect of PR-A and PR-B alone and in combination on progesterone-induced PRE-Luc activity. A, Western blot of lysates (50 µg/lane) from transfected PHM1–31 cells probed simultaneously with PgR1294 and anti-GAPDH. B, Effect of progesterone (100 nM for 8 h) or vehicle on relative PRE-Luc activity in PHM1–31 cells transfected with empty plasmid or PR-A and PR-B expression plasmids.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
We tested the hypothesis that human parturition involves specific changes in myometrial nPR expression that could mediate functional progesterone withdrawal. Our previous qRT-PCR data (13), and the Western blot analyses reported by Pieber et al. (14) supported this hypothesis and indicated that the onset of labor is associated with increased PR-A expression. However, the methods used to measure PR-A expression were equivocal. More recently Condon et al. (15) reported that PR-A is not expressed by human pregnancy myometrium at term. Instead, their Western blot assay indicated that PR-C increase with labor onset and only in myometrium from the uterine fundus. They proposed that an increase in PR-C, and not PR-A, mediates functional progesterone withdrawal and that the human pregnancy myometrium is regionalized such that functional progesterone withdrawal occurs only in the fundus. Although those findings contradict our earlier data, the central premise that functional progesterone withdrawal involves specific changes in myometrial nPR expression is consistent. However, the study by Condon et al. (15) also had technical limitations. In particular, the identities of the putative nPR proteins, especially the band designated to represent PR-C, are uncertain.

To resolve these technical issues, we developed and validated nPR Western blot and IHC assays based on the PgR1294 monoclonal nPR antibody. This antibody targets the hinge region between amino acids 400 and 534 of the human nPR protein and therefore detects PR-A and PR-B but not PR-C (22). Our PgR1294-Western blot assay was highly specific for PR-A and PR-B and readily detected those proteins in myometrial lysates. We found that PR-A and PR-B proteins were present in the lower segment of the human pregnancy uterus and localized exclusively to the nucleus of myometrial cells. Consistent with our previous mRNA data, we found that PR-A levels in lower segment myometrium increased with advancing gestation and in association with the onset of labor, whereas PR-B levels remained constant during the final weeks of the third trimester and at parturition. The specific increase in PR-A expression resulted in a significant pregnancy stage- and labor-associated increase in the myometrial PR-A to PR-B protein ratio. In preterm specimens, the PR-A to PR-B protein ratio was around 0.5 and favored PR-B; by term the ratio doubled to be around 1; and in laboring myometrium it doubled further to favor PR-A by a 2.5:1 ratio. These findings are consistent with earlier studies (13, 14) and support the hypothesis that human parturition involves a specific increase in the myometrial PR-A to PR-B ratio due to increased expression of PR-A. Interestingly, in the rhesus macaque, a species that also lacks a systemic progesterone withdrawal at parturition, myometrial PR-A protein levels and the PR-A to PR-B protein ratio increase during late gestation and in association with labor onset (23) in a manner practically identical with our current data, suggesting that the increase in myometrial PR-A expression is a conserved trait in primate parturition. In both species myometrial nPR levels were relatively low, consistent with the fact that the myometrium is exposed to high levels of progesterone, which represses nPR expression (24, 25, 26). The difference between our data and those of Condon et al. (15), who failed to detect PR-A in the pregnancy myometrium, is likely due to differences in nPR antibodies. However, our data do not dispute the findings of Condon et al., indicating that PR-C is expressed in the fundal myometrium because we did not examine fundal specimens, and in any case, our nPR assays could not detect PR-C.

The idea that fundal and lower segment myometrium differ functionally is controversial. It is thought that during labor the fundus contracts and the lower segment relaxes. Indeed the data of Condon et al. (15) suggesting that functional progesterone withdrawal occurs only in fundal myometrium support this hypothesis. However, our present data clearly show that PR-A is expressed in lower segment myometrium and that it increases with advancing pregnancy and labor onset. Thus, functional progesterone withdrawal may occur in the lower uterine segment. It is possible that reported differences between fundal and lower segment myometrium are simply due to differences in tissue composition, i.e. the fundus may have a higher myometrial cell density than the lower uterine segment, which is closer to the myometrium/cervix interface. This is exemplified by our IHC data showing that myometrial specimens contain an inordinate and variable amount of nonmyometrial tissue. To account for this variability, we used h-CAL as a normalizing factor for nPR protein levels because both genes are expressed exclusively in myometrial cells, and h-CAL expression is constitutive and not affected by stage of pregnancy or labor status (we confirmed this by qRT-PCR analyses; data not shown). Thus, the notion that myometrium in the fundus is phenoptypically different to myometrium in the lower uterine segment remains a contentious issue.

Transition of the pregnancy myometrium from a quiescent to a contractile state was associated with an increase in the myometrial PR-A to PR-B protein ratio. Our earlier study indicated that myometrial progesterone responsiveness (based on the extent of endogenous progesterone responsive gene expression) decreases progressively before the onset of active labor and is inversely related to the PR-A to PR-B mRNA ratio (13). The present data are consistent with those findings and support the concept that the process of human parturition begins before the onset of active labor and involves the specific increase in myometrial PR-A expression. Further studies addressing the regulation of myometrial PR-A expression may elucidate key aspects of the hormonal control of human parturition. We previously showed that in PHM1–31 cells prostaglandin F2{alpha} (PGF2{alpha}) stimulates PR-A but not PR-B expression (27). We proposed that increased PGF2{alpha} production by the gestational tissues induces functional progesterone withdrawal by increasing myometrial PR-A expression. This model directly links progesterone withdrawal with a key immune/inflammatory modulator at the maternal/fetal interface and explains how administration of PGF2{alpha} induces the full parturition cascade.

The induction of labor by the nPR antagonist RU486 indicates that progesterone promotes myometrial relaxation and/or blocks labor onset primarily via a genomic mode of action. Although the existence of specific membrane-associated PRs in the pregnancy myometrium (28, 29, 30) helps explain the well-documented rapid relaxatory effects of progesterone on myometrial contractility (31), the involvement of this mode of progesterone action in the control of human pregnancy and parturition is unclear, especially because RU486 is not thought to inhibit membrane-associated PR function. The possibility that PR-A acts as an nPR antagonist is important in this regard. We propose that progesterone maintains a relaxed myometrium and blocks labor via its interaction with PR-B. Our studies in PHM1–31 cells show that genomic progesterone actions (at least at the classic progesterone response element) are mediated by PR-B and not by PR-A. The PR-A to PR-B ratio favored PR-B by a 2:1 margin in relaxed preterm specimens. The PR-A to PR-B ratio increased between preterm and term specimens, indicating that the trajectory of myometrial genomic progesterone responsiveness changes toward a decrease with advancing gestation. We speculate that the PR-A to PR-B ratio of around 1 in nonlaboring term myometrium was close to the threshold for functional progesterone withdrawal. In laboring term myometrium, the PR-A to PR-B protein ratio was greater than 1 and more than double nonlaboring levels. We do not know whether this increase occurred before or after labor onset. We speculate that the PR-A to PR-B ratio continues to increase until the threshold for functional progesterone withdrawal is reached. In breast tumor cell lines, repressive actions of PR-A on PR-B activity occur when the PR-A to PR-B ratio exceeds unity (32). We found that repression of PR-B activity by PR-A in PHM1–31 cells occurred at PR-A to PR-B levels within the range measured in term laboring myometrium. Thus, the PR-A to PR-B ratio in laboring term myometrium likely was sufficient to produce functional progesterone withdrawal. However, whether the labor-associated increase in the PR-A to PR-B ratio was causal to labor or consequential to labor onset remains uncertain.

Several studies have shown that PR-A mediates distinct (compared with PR-B) actions of progesterone (33, 34). PR-B-knockout mice have a normal pregnancy and parturition, indicating that myometrial expression of PR-A alone is sufficient for progesterone to maintain pregnancy and promote myometrial relaxation (35). In contrast, mice lacking PR-A have ovarian deficiencies and embryos fail to implant (36). Thus, it is possible that ligand-activated PR-A modulates expression of specific genes and that the increase in myometrial PR-A expression confers a distinct set of progesterone actions on the pregnancy myometrium. Notwithstanding this possibility, our data show that human parturition involves a specific increase in the myometrial PR-A expression, which increases the PR-A to PR-B protein ratio, and we propose that at least one consequence of this is functional progesterone withdrawal due to PR-A-mediated inhibition of PR-B activity.


   Acknowledgments
 
We thank Dr. Neal Rote for critically assessing the manuscript.


   Footnotes
 
This work was supported by Grant HD051563-01 from the National Institutes of Health and March of Dimes Birth Defects Foundation Grant 6-FY05-68, and the Departments of Obstetrics and Gynecology, University Hospitals, Case Medical Center and Reproductive Biology, Case Western Reserve University.

All authors have nothing to declare regarding potential conflicts of interest.

First Published Online March 6, 2007

Abbreviations: GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; h-CAL, high molecular weight caldesmon; IHC, immunohistochemistry; nPR, nuclear PR; PGF2{alpha}, prostaglandin F2{alpha}; PR, progesterone receptor; PTNIL, cesarean delivery preterm, not in labor, and requiring cesarean delivery; qRT-PCR, quantitative RT-PCR; siRNA, small interfering RNA; TIL, at term and in labor; TNIL, at term and not in labor; TTBS, Tris, NaCl, and Tween 20.

Received January 12, 2007.

Accepted February 27, 2007.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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