The Progesterone Receptor in Human Term Amniochorion and Placenta Is Isoform C
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《内分泌学杂志》
Preterm Birth Research Group, Reproductive Sciences, Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester, Leicestershire LE2 7LX, United Kingdom
Abstract
The mechanism that initiates human parturition has been proposed to be functional progesterone withdrawal whereby the 116-kDa B isoform of the progesterone receptor (PR-B) switches in favor of the 94-kDa A isoform (PR-A) in reproductive tissues. Recently other PR isoforms, PR-S, PR-C, and PR-M generated from the same gene have been identified and partially characterized. Using immunohistochemical, Western blotting, and RT-PCR techniques, evidence is provided that the major PR isoform present in human term fetal membranes (amnion and chorion) and syncytiotrophoblast of the placenta is neither of the classical nuclear PR-B or PR-A isoforms but is the N terminally truncated 60-kDa PR-C isoform. Evidence is also provided that the PR-C isoform resides in the cytoplasm of the expressing cell types. Data are also presented to show that PR-B, PR-A, and PR-S isoforms are essentially absent from the amnion and chorion, whereas PR isoforms A, B, C, and S are all present in the decidua, with PR-A being the major isoform. The syncytiotrophoblast of the placenta contains the cytoplasmic PR-C isoform but not PR-A, PR-B, or PR-S. The major PR isoform in the amnion, chorion, and placenta is PR-C, suggesting that the cytoplasmic PR-C isoform has a specific role in extraembryonic tissues and may be involved in the regulation of human parturition.
Introduction
PROGESTERONE RECEPTORS (PRs) ARE members of a superfamily of ligand-activated nuclear transcription factors comprised of specific domains involved in DNA binding, hormone binding, and transactivation (1). Progesterone activation of PR in target tissues is mediated via dimerization and phosphorylation of the receptor, resulting in binding to cis-acting progesterone response elements on DNA and the modulation of the promoters of target genes (1, 2). The human PR-A isoform differs from the PR-B isoform in lacking the first 164 amino acids contained in PR-B (3). Both are translated from distinct mRNA transcripts generated from a single gene under the control of separate estrogen sensitive promoters (4). Previous work (5) has identified three additional AUG translation sites with a possible methionine site at 595 that is predicted to generate a protein of approximately 60 kDa.
Although much work has been performed on PR-B and PR-A, little work has been undertaken on the other seven transcripts generated from the PR gene (5), despite there being evidence that some of these are translated into functional 38-, 60-, 71, or 78-kDa proteins in malignant progesterone target tissues (6) and that these are coordinately up-regulated by estrogens and down-regulated by progestins (7, 8). Evidence also exists for other PR isoform such as PR-C, PR-S, and PR-T, which could be genomic mediators of progestin action (9, 10), and for three membrane progestin receptors that are classical G-coupled protein receptor transduction molecules first identified in the teleost oocyte called mPR, mPR, and mPR (11, 12).
PRs have been proposed to play a key role in the control of human labor and parturition whereby the levels of the PR-B isoform, which is often considered to be the dominant isoform, fall during labor, leaving the PR-A isoform as the predominant form leading to a functional PR withdrawal (13). Evidence to support this occurring in the uterine myometrium exists (14). In other human reproductive tissues, such as the decidua, ovary, and oviduct (15, 16), PR-A appears to be the predominant progestin regulator, with PR-B maintaining a supporting role, suggesting that progestin signaling in the human uterus at the end of parturition is far more complex than a PR-B to PR-A isoform switching mechanism (14). Despite there being a paucity of data to support functional PR withdrawal in tissues at the fetal-maternal interface, i.e. in the fetal membranes, decidua, and placenta, many still consider that only the PR-B and PR-A isoforms are present (17, 18).
Recent data have suggested that at least five nuclear PR isoforms are present in the human decidua and that all five isoforms are decreased after labor (19). However, although Western blotting techniques also indicated the presence of several PR isoforms in amniotic nuclear extracts, immunohistochemical methods failed to detect any PR isoforms in the amnion and chorion (19).
In the present study examining the pattern of expression of PR isoforms in human fetal membranes (amnion and chorion), decidua, and placenta at term, we demonstrate that the major isoform present in the fetal membranes and placenta is a cytoplasmic 60-kDa PR-C isoform, that PR-B or PR-A is not expressed in the amnion or chorion, and the 94-kDa PR-A protein is the dominant PR isoform in the decidua.
Materials and Methods
Patient samples
Local research ethics committee approval for the study was obtained and all patients signed informed consent for their tissues to be used. Fetal membranes and placenta (n = 6) were collected from term patients undergoing elective cesarean section before labor. All tissues were divided into three parts: one fixed in formalin and embedded in paraffin for histological examination and the other two snap frozen in liquid nitrogen and stored at –80 C for later RNA and protein extraction. Enriched amnion was obtained by carefully peeling the amnion from chorion and decidua. Enriched decidua was obtained using the edge of a microscope slide to carefully remove a thin layer from the inverted fetal membranes. Enriched chorion was obtained by using the edge of the microscope slide to scrape away the remaining decidua. A 2-cm3 block of placenta from the midpart of a cotyledon was taken and washed briefly with sterile PBS before division into three parts.
Immunohistochemistry
Five-micrometer sections of tissue dried for 48 h before dewaxing and rehydration through graded alcohol to H2O were subjected to boiling 10 mM citric acid for 12 min followed by cooling for exactly 20 min before transfer into cold H2O to retrieve antigenic sites. Endogenous peroxidase activity and nonspecific binding was blocked with 6% H2O2 for 15 min, followed by incubation with nonimmune rabbit serum (10% in PBS) for 30 min, respectively. Endogenous avidin and biotin sites were blocked with avidin-binding blocking solutions (Vector Laboratories, Peterborough, UK) according to the manufacturer’s instructions. Primary antibody, PR clones 1A6, San27, clone 16, or the polyclonal antibody C-20 (SC-539; Santa Cruz Biotechnology, Santa Cruz, CA) were applied at the indicated concentrations (Fig. 1) in 10% nonimmune rabbit serum or 10% swine serum overnight at 4 C. In some studies, C-20 antibodies were preincubated (3 h at room temperature) with a 7-fold excess of immunizing peptide before use. After copious washing in PBS, biotinylated rabbit antimouse or swine antirabbit secondary antibodies (1:400) were applied for 1 h 30 min, the sections washed, and avidin-biotin complexes applied for 30 min. Color was developed over antigenic sites using 3, 3'-diaminobenzidine for 5 min. After copious washes in dH2O, sections were lightly counterstained with hematoxylin, dehydrated through graded alcohols, cleared in xylene, and permanently mounted in XAM (BDH, Poole, UK). Photomicrographs were obtained at the indicated magnifications on a Axioplan compound microscope (Zeiss, Welwyn Garden City, UK) fitted with a DN-100 digital camera (Nikon, Kingston upon Thames, UK). Images were digitally enhanced using proprietary software.
RNA preparation and RT-PCR
Total cellular RNA was obtained from 100 mg of tissue using Trizol reagent (Invitrogen, Paisley, UK) according the manufacturer’s instructions and genomic DNA contamination removed by treating the samples with RNase-free DNase 1 (Promega, Southampton, UK) for 1 h at 37 C, followed by phenol-chloroform extraction and isopropanol precipitation. After verification of RNA quality by UV-spectrophotometer analysis and agarose gel electrophoresis, 1 μg of RNA was reverse transcribed using avian myeloblastosis virus (AMV)-reverse transcriptase (RT) for 1 h at 42 C. A minus RT control was obtained by substituting diethylpyrocarbonate-treated water for the AMV-RT enzyme.
PCR was performed using PR primer combinations that identified all PR isoforms, the PR-B isoform, PR-A and PR-B isoforms, or PR-S isoform (20) (Table 1).
Western blotting
Protein extracts were obtained by homogenizing samples in ice-cold lysis buffer [1% Ipegcal, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 2 mM EDTA, 50 mM sodium fluoride, 1 mM sodium metavanadate, 0.2 mg/ml aprotinin, all from Sigma-Aldrich, Poole UK, in PBS (pH 7.0) but without phenyl methyl sulfonyl fluroride] (21). After incubation on ice for 30 min, insoluble material was removed by centrifugation at 14,000 x g for 20 min at 4 C and supernatants stored at –20 C. One hundred micrograms of protein per lane were resolved by 7.5% SDS-PAGE, transferred to nitrocellulose membranes (Amersham-Pharmacia Biotech, Chalfont St. Giles, UK), and probed with anti-PR antibodies 1:50 (clone 1A6) or 1:400 (C-20) in 3% Blotto. Peroxidase-conjugated secondary antibody (Amersham) was used at a 1:2000 dilution and the reaction visualized using enhanced chemiluminescence detection kits. Negative controls were performed with blots not exposed to primary antibody and by incubation of antibodies with a 7-fold excess of immunizing peptide or extracts produced from choriocarcinoma (BeWo) cells transiently transfected with human PR-A (22) or PR-C (9) expression plasmids.
Results
To investigate the PR isoform repertoire of the progesterone-dependent human fetal-maternal interface, immunohistochemical analysis using the commercial PR antibody clones 1A6 and C-20 generated toward the ligand- and DNA-binding domains, respectively, of the full-length PR-B isoform were used (Fig 1). The data revealed the presence of cytoplasmic staining in the amnion epithelial cell, chorionic trophoblast, and maternal decidual cell. Nuclear staining with these antibodies was found only in decidual cells and positive control tissues such as breast cancer, endometrium, and myometrium (positive control data not shown). No nuclear staining was detected in the amnion epithelium and chorionic cytotrophoblast (Fig. 1). The connective tissue cells of the reticular and chorionic layers of the fetal membrane were devoid of any immunoreactivity (Fig. 1A). In the placenta, the syncytiotrophoblast also demonstrated cytoplasmic staining with this antibody and no nuclear staining (Fig. 1C). However, cytoplasmic and nuclear immunoreactivity was observed in decidual cells attached to the basal plate (Fig. 1C).
To confirm the identity of the cytoplasmic isoform found in these cell types, immunostaining with PR-B- and PR-A-specific antibodies revealed nuclear staining in the decidual cell but no other staining elsewhere in the fetal membrane (Fig. 1). Similarly, nuclear staining was confined only to the decidual cells of the basal plate attached to the placenta (Fig. 1E). Nuclear staining in classical progesterone target tissues (breast cancer, endometrial, and myometrial cells) confirmed the PR-B and PR-A specificity of the antibodies (data not shown). The conclusion from these studies was that the cytoplasmic isoform found within fetal membranes, decidua, and syncytiotrophoblast of the placenta was neither the PR-B nor the PR-A isoform and suggestive of one of the other PR isoforms. These data were inconsistent with previous observations that the amnion epithelial cell contained both PR-B and PR-A isoforms and that during labor there is a loss of the PR-B isoform in favor of the PR-A isoform (17) but is consistent with immunohistochemical studies of Goldman et al. (19).
To find the identity of the cytoplasmic PR isoform, RNA extracts prepared from term fetal membrane and placenta samples were subjected to RT-PCR with several isoform-specific primer sets (Table 1) and revealed the presence of a PR transcript in all samples by RT-PCR (Fig. 2, A and B, top panels). Analysis with PR-B-, PR-A/PR-B-, and PR-S-specific primer sets showed that fetal membranes with decidua attached consisted mainly of PR-A transcripts with some transcripts for PR-B but no PR-S transcripts (Fig. 2). Fetal membranes enriched for amniochorion revealed the presence of a transcript for PR that was not PR-B, PR-A, or PR-S, leading to conclusion that the cytoplasmic PR isoform present was either PR-C or PR-M previously observed in human breast cancer cells and isolated from human aortic endothelial cells, respectively (6, 9, 23). The placenta similarly contained a transcript for PR that was either PR-C or PR-M (Fig. 2B). Samples enriched for decidua revealed the presence of all PR isoforms including PR-S (Fig. 2B).
When protein extracts from partially purified fetal membranes, placenta, trophoblasts, and decidua were compared with those from T47D breast cancer cells, using Western blotting techniques with the PR antibodies that detected the cytoplasmic isoform in the immunohistochemistry studies (Fig. 1 and Table 2), several PR-immunoreactive proteins were observed in T47D breast cancer cell extracts in line with previous studies (8, 19, 21), whereas the tissues at the fetal maternal interface revealed a major PR isoform with a relative molecular mass of approximately 60 kDa (Fig. 3). This isoform was confirmed as the PR-C isoform, as previously reported in human breast cancer cells (5, 23), human fetal membranes (19), and the guinea pig cervix (21), with recombinant protein and preadsorption studies (Fig. 3). Other progesterone target tissues, such as endometrium (data not shown) and myometrium, possessed the 116-kDa PR-B, 94-kDa PR-A, and 60-kDa PR-C isoforms but only with the C-20 antibody (Fig. 3).
Discussion
The role of PRs in human gestation and parturition is to maintain pregnancy. In most mammals, the stability of the relationship in the fetal-maternal interface is disturbed by the fall of progesterone production by the placenta, with the concomitant softening of the cervix, rupture of the fetal membranes, and initiation of highly synchronized high-pressure contractions of the myometrium that characterizes labor (24). In the human, similar events occur in the same coordinated manner except there is no loss of systemic progesterone (24). A hypothesis presented (17), that a switch in PR isoforms from the more transcriptionally dominant PR-B to the less active PR-A isoform must therefore occur in these tissues, has been convincingly supported by evidence in the myometrium (25, 26) and cervix (27, 28) and less convincingly in the amnion and maternal decidua (19).
In the present study, we have shown that the fetal membranes (amnion epithelial cell and chorionic cytotrophoblast) contain mainly high levels of PR-C (Fig. 1 and Table 2), whereas the placental cytotrophoblast is devoid of PR and the placental syncytiotrophoblast contains both PR-C and PR-A (Table 2). By contrast, the maternal decidua contains not only PR-A, as its major isoform, but also PR-B, PR-S, and the cytoplasmic PR-C. These data differ from those recently reported (19) in which PR-B and PR-A isoforms were observed in amnion nuclear extracts, yet paradoxically the antibody used failed to show immunoreactive PR by immunohistochemistry and theoretically should not detect the PR-C, PR-S, or PR-M isoforms in Western blotting because the epitope for this antibody is found only at the N terminal region of the PR-A isoform (29). In the present study, no PR-B or PR-A isoforms were detected in the amnion or chorion and the hypothesis that progesterone receptor switching occurs within these tissues at term or in labor (17) is not supported. Although we cannot rule out the presence of a small amount of PR-B or PR-A isoforms being present in the nuclei of amnion epithelial cells, despite a lack of expression of these isoforms at the mRNA level (Fig. 2), the present study indicates that PR-B and PR-A are of little importance in relation to PR-C in fetal membranes. Additionally, the PR immunoreactivity in the present study that we consider represents the C isoform was confined to the cytoplasm of the amniotic epithelium and chorionic cytotrophoblast and was the major isoform present, whereas no cytoplasmic immunoreactivity was detected in the aforementioned study (19).
These data are qualitatively similar to that obtained in the baboon (30), in which PR was not observed in the amnion or chorion, but strong nuclear and weak cytoplasmic staining was observed in the decidua with the JZB39 rat anti-PR monoclonal antibody. Observations obtained with JZB39 (31), which has been shown to detect only the PR-C isoform in myometrium and not T47D extracts by immunoblotting, were dependent on protein concentrations (31). These data suggest that protein loading in immunoblotting is critical for good PR-C detection. Indeed, PR-C was weakly represented on Western blotting in a previous study (19), whereas our data show intense PR-C expression (Fig. 3) for this exact reason. Alternatively, these discrepant observations may be related to the antibodies used; the use of nuclear extracts compared with whole cell extracts; or the presence of contaminating cytoplasm/tissues in the previous study (19).
In agreement with our studies, Goldman et al. (19) showed that PR-A is expressed in the nucleus of the decidua but absent in the amnion by immunohistochemistry but paradoxically found all isoforms present by Western blotting. By contrast, our Western blot studies indicate the major PR isoform in the fetal membranes (amnion and chorion) is C and that it is localized within the cytoplasm, without immunoreactivity within the nuclei of the same cells, and, therefore, we consider that the relevance of the changes in levels of the PR isoforms detected within the amniotic nuclear extracts of labor (19) should be considered with caution.
Using antibodies that detect all known PR isoforms, we demonstrated that the major isoform in the amnion, chorion, and syncytiotrophoblast is the 60-kDa PR-C isoform (Fig. 3). The absence of PR-B and PR-A transcripts in these tissues (Fig. 2) confirmed these findings. By contrast, the major isoform in the term decidua is PR-A, but PR-B, PR-S, and PR-C isoforms are also expressed. In light of the study by Goldman et al., these data need further clarification but suggest that PR-C is an important molecule in the fetal membranes, decidua, and syncytiotrophoblast. These data also suggest that a reevaluation of the roles of PR in the fetal-maternal interface is needed, and the idea that PR-A simply acts as a dominant-negative regulator of PR-B action is not applicable to these tissues (17).
The mechanism involved in the synthesis of the PR-C isoform is unknown, but limited evidence suggests that it may be produced using a third estrogen-dependent promoter with an AUG at amino acid 595 being the translational start site (5). The demonstration that PR-C isoforms in the human breast cancer cell do not arise from proteolysis of larger PR isoforms (9) and that specific PR-C mRNA transcripts are present in human fetal membranes and placenta (this study) strongly suggests that the PR-C isoform is generated through promoter-specific transcription. The function of PR-C is unclear, although it may act as modulator of PR-A and PR-B transcriptional activities in those cells that produce these isoforms (9). In the human amnion and chorion, dominant-negative regulation of PR-B and PR-A transcriptional control seems unlikely in the absence of any measurable PR-A or PR-B isoforms and points toward PR-C-specific functions. Because PR-C lacks a full DNA binding domain (9) but has a nuclear localization signal and two dimerization domains, the intriguing possibility that PR-C may associate with other transcriptional elements to modulate gene transcription is raised. However, the cytoplasmic location and relatively high levels of this protein are suggestive of a localized function in the cytoplasm rather than a nuclear genomic function and that this localization is important for sustained gestation. It now seems imperative that the regulation and role of the PR-C isoform is investigated to further our understanding of the role of progesterone in these tissues during human parturition.
Acknowledgments
The authors thank Reshma Bharkhada for excellent technical assistance and Dr. O. Habayeb and Mr. M. Habiba (University Hospitals Leicester) and Prof. L. Jones (St. Bartholemew’s Hospital, London, UK) for assistance in the procurement of clinical specimens and tissue extracts. The human PR-A and PR-C expression plasmids were kind gifts of Dr. Christine Clarke (Westmead Institute for Cancer Research, University of Sydney, Australia) and Dr. Kathryn Horwitz (University of Colorado Health Sciences Center, Denver, CO), respectively.
Footnotes
The authors have no conflicts of interest.
First Published Online October 27, 2005
Abbreviations: AMV, Avian myeloblastosis virus; PR, progesterone receptor; RT, reverse transcriptase.
Accepted for publication October 19, 2005.
References
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Abstract
The mechanism that initiates human parturition has been proposed to be functional progesterone withdrawal whereby the 116-kDa B isoform of the progesterone receptor (PR-B) switches in favor of the 94-kDa A isoform (PR-A) in reproductive tissues. Recently other PR isoforms, PR-S, PR-C, and PR-M generated from the same gene have been identified and partially characterized. Using immunohistochemical, Western blotting, and RT-PCR techniques, evidence is provided that the major PR isoform present in human term fetal membranes (amnion and chorion) and syncytiotrophoblast of the placenta is neither of the classical nuclear PR-B or PR-A isoforms but is the N terminally truncated 60-kDa PR-C isoform. Evidence is also provided that the PR-C isoform resides in the cytoplasm of the expressing cell types. Data are also presented to show that PR-B, PR-A, and PR-S isoforms are essentially absent from the amnion and chorion, whereas PR isoforms A, B, C, and S are all present in the decidua, with PR-A being the major isoform. The syncytiotrophoblast of the placenta contains the cytoplasmic PR-C isoform but not PR-A, PR-B, or PR-S. The major PR isoform in the amnion, chorion, and placenta is PR-C, suggesting that the cytoplasmic PR-C isoform has a specific role in extraembryonic tissues and may be involved in the regulation of human parturition.
Introduction
PROGESTERONE RECEPTORS (PRs) ARE members of a superfamily of ligand-activated nuclear transcription factors comprised of specific domains involved in DNA binding, hormone binding, and transactivation (1). Progesterone activation of PR in target tissues is mediated via dimerization and phosphorylation of the receptor, resulting in binding to cis-acting progesterone response elements on DNA and the modulation of the promoters of target genes (1, 2). The human PR-A isoform differs from the PR-B isoform in lacking the first 164 amino acids contained in PR-B (3). Both are translated from distinct mRNA transcripts generated from a single gene under the control of separate estrogen sensitive promoters (4). Previous work (5) has identified three additional AUG translation sites with a possible methionine site at 595 that is predicted to generate a protein of approximately 60 kDa.
Although much work has been performed on PR-B and PR-A, little work has been undertaken on the other seven transcripts generated from the PR gene (5), despite there being evidence that some of these are translated into functional 38-, 60-, 71, or 78-kDa proteins in malignant progesterone target tissues (6) and that these are coordinately up-regulated by estrogens and down-regulated by progestins (7, 8). Evidence also exists for other PR isoform such as PR-C, PR-S, and PR-T, which could be genomic mediators of progestin action (9, 10), and for three membrane progestin receptors that are classical G-coupled protein receptor transduction molecules first identified in the teleost oocyte called mPR, mPR, and mPR (11, 12).
PRs have been proposed to play a key role in the control of human labor and parturition whereby the levels of the PR-B isoform, which is often considered to be the dominant isoform, fall during labor, leaving the PR-A isoform as the predominant form leading to a functional PR withdrawal (13). Evidence to support this occurring in the uterine myometrium exists (14). In other human reproductive tissues, such as the decidua, ovary, and oviduct (15, 16), PR-A appears to be the predominant progestin regulator, with PR-B maintaining a supporting role, suggesting that progestin signaling in the human uterus at the end of parturition is far more complex than a PR-B to PR-A isoform switching mechanism (14). Despite there being a paucity of data to support functional PR withdrawal in tissues at the fetal-maternal interface, i.e. in the fetal membranes, decidua, and placenta, many still consider that only the PR-B and PR-A isoforms are present (17, 18).
Recent data have suggested that at least five nuclear PR isoforms are present in the human decidua and that all five isoforms are decreased after labor (19). However, although Western blotting techniques also indicated the presence of several PR isoforms in amniotic nuclear extracts, immunohistochemical methods failed to detect any PR isoforms in the amnion and chorion (19).
In the present study examining the pattern of expression of PR isoforms in human fetal membranes (amnion and chorion), decidua, and placenta at term, we demonstrate that the major isoform present in the fetal membranes and placenta is a cytoplasmic 60-kDa PR-C isoform, that PR-B or PR-A is not expressed in the amnion or chorion, and the 94-kDa PR-A protein is the dominant PR isoform in the decidua.
Materials and Methods
Patient samples
Local research ethics committee approval for the study was obtained and all patients signed informed consent for their tissues to be used. Fetal membranes and placenta (n = 6) were collected from term patients undergoing elective cesarean section before labor. All tissues were divided into three parts: one fixed in formalin and embedded in paraffin for histological examination and the other two snap frozen in liquid nitrogen and stored at –80 C for later RNA and protein extraction. Enriched amnion was obtained by carefully peeling the amnion from chorion and decidua. Enriched decidua was obtained using the edge of a microscope slide to carefully remove a thin layer from the inverted fetal membranes. Enriched chorion was obtained by using the edge of the microscope slide to scrape away the remaining decidua. A 2-cm3 block of placenta from the midpart of a cotyledon was taken and washed briefly with sterile PBS before division into three parts.
Immunohistochemistry
Five-micrometer sections of tissue dried for 48 h before dewaxing and rehydration through graded alcohol to H2O were subjected to boiling 10 mM citric acid for 12 min followed by cooling for exactly 20 min before transfer into cold H2O to retrieve antigenic sites. Endogenous peroxidase activity and nonspecific binding was blocked with 6% H2O2 for 15 min, followed by incubation with nonimmune rabbit serum (10% in PBS) for 30 min, respectively. Endogenous avidin and biotin sites were blocked with avidin-binding blocking solutions (Vector Laboratories, Peterborough, UK) according to the manufacturer’s instructions. Primary antibody, PR clones 1A6, San27, clone 16, or the polyclonal antibody C-20 (SC-539; Santa Cruz Biotechnology, Santa Cruz, CA) were applied at the indicated concentrations (Fig. 1) in 10% nonimmune rabbit serum or 10% swine serum overnight at 4 C. In some studies, C-20 antibodies were preincubated (3 h at room temperature) with a 7-fold excess of immunizing peptide before use. After copious washing in PBS, biotinylated rabbit antimouse or swine antirabbit secondary antibodies (1:400) were applied for 1 h 30 min, the sections washed, and avidin-biotin complexes applied for 30 min. Color was developed over antigenic sites using 3, 3'-diaminobenzidine for 5 min. After copious washes in dH2O, sections were lightly counterstained with hematoxylin, dehydrated through graded alcohols, cleared in xylene, and permanently mounted in XAM (BDH, Poole, UK). Photomicrographs were obtained at the indicated magnifications on a Axioplan compound microscope (Zeiss, Welwyn Garden City, UK) fitted with a DN-100 digital camera (Nikon, Kingston upon Thames, UK). Images were digitally enhanced using proprietary software.
RNA preparation and RT-PCR
Total cellular RNA was obtained from 100 mg of tissue using Trizol reagent (Invitrogen, Paisley, UK) according the manufacturer’s instructions and genomic DNA contamination removed by treating the samples with RNase-free DNase 1 (Promega, Southampton, UK) for 1 h at 37 C, followed by phenol-chloroform extraction and isopropanol precipitation. After verification of RNA quality by UV-spectrophotometer analysis and agarose gel electrophoresis, 1 μg of RNA was reverse transcribed using avian myeloblastosis virus (AMV)-reverse transcriptase (RT) for 1 h at 42 C. A minus RT control was obtained by substituting diethylpyrocarbonate-treated water for the AMV-RT enzyme.
PCR was performed using PR primer combinations that identified all PR isoforms, the PR-B isoform, PR-A and PR-B isoforms, or PR-S isoform (20) (Table 1).
Western blotting
Protein extracts were obtained by homogenizing samples in ice-cold lysis buffer [1% Ipegcal, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 2 mM EDTA, 50 mM sodium fluoride, 1 mM sodium metavanadate, 0.2 mg/ml aprotinin, all from Sigma-Aldrich, Poole UK, in PBS (pH 7.0) but without phenyl methyl sulfonyl fluroride] (21). After incubation on ice for 30 min, insoluble material was removed by centrifugation at 14,000 x g for 20 min at 4 C and supernatants stored at –20 C. One hundred micrograms of protein per lane were resolved by 7.5% SDS-PAGE, transferred to nitrocellulose membranes (Amersham-Pharmacia Biotech, Chalfont St. Giles, UK), and probed with anti-PR antibodies 1:50 (clone 1A6) or 1:400 (C-20) in 3% Blotto. Peroxidase-conjugated secondary antibody (Amersham) was used at a 1:2000 dilution and the reaction visualized using enhanced chemiluminescence detection kits. Negative controls were performed with blots not exposed to primary antibody and by incubation of antibodies with a 7-fold excess of immunizing peptide or extracts produced from choriocarcinoma (BeWo) cells transiently transfected with human PR-A (22) or PR-C (9) expression plasmids.
Results
To investigate the PR isoform repertoire of the progesterone-dependent human fetal-maternal interface, immunohistochemical analysis using the commercial PR antibody clones 1A6 and C-20 generated toward the ligand- and DNA-binding domains, respectively, of the full-length PR-B isoform were used (Fig 1). The data revealed the presence of cytoplasmic staining in the amnion epithelial cell, chorionic trophoblast, and maternal decidual cell. Nuclear staining with these antibodies was found only in decidual cells and positive control tissues such as breast cancer, endometrium, and myometrium (positive control data not shown). No nuclear staining was detected in the amnion epithelium and chorionic cytotrophoblast (Fig. 1). The connective tissue cells of the reticular and chorionic layers of the fetal membrane were devoid of any immunoreactivity (Fig. 1A). In the placenta, the syncytiotrophoblast also demonstrated cytoplasmic staining with this antibody and no nuclear staining (Fig. 1C). However, cytoplasmic and nuclear immunoreactivity was observed in decidual cells attached to the basal plate (Fig. 1C).
To confirm the identity of the cytoplasmic isoform found in these cell types, immunostaining with PR-B- and PR-A-specific antibodies revealed nuclear staining in the decidual cell but no other staining elsewhere in the fetal membrane (Fig. 1). Similarly, nuclear staining was confined only to the decidual cells of the basal plate attached to the placenta (Fig. 1E). Nuclear staining in classical progesterone target tissues (breast cancer, endometrial, and myometrial cells) confirmed the PR-B and PR-A specificity of the antibodies (data not shown). The conclusion from these studies was that the cytoplasmic isoform found within fetal membranes, decidua, and syncytiotrophoblast of the placenta was neither the PR-B nor the PR-A isoform and suggestive of one of the other PR isoforms. These data were inconsistent with previous observations that the amnion epithelial cell contained both PR-B and PR-A isoforms and that during labor there is a loss of the PR-B isoform in favor of the PR-A isoform (17) but is consistent with immunohistochemical studies of Goldman et al. (19).
To find the identity of the cytoplasmic PR isoform, RNA extracts prepared from term fetal membrane and placenta samples were subjected to RT-PCR with several isoform-specific primer sets (Table 1) and revealed the presence of a PR transcript in all samples by RT-PCR (Fig. 2, A and B, top panels). Analysis with PR-B-, PR-A/PR-B-, and PR-S-specific primer sets showed that fetal membranes with decidua attached consisted mainly of PR-A transcripts with some transcripts for PR-B but no PR-S transcripts (Fig. 2). Fetal membranes enriched for amniochorion revealed the presence of a transcript for PR that was not PR-B, PR-A, or PR-S, leading to conclusion that the cytoplasmic PR isoform present was either PR-C or PR-M previously observed in human breast cancer cells and isolated from human aortic endothelial cells, respectively (6, 9, 23). The placenta similarly contained a transcript for PR that was either PR-C or PR-M (Fig. 2B). Samples enriched for decidua revealed the presence of all PR isoforms including PR-S (Fig. 2B).
When protein extracts from partially purified fetal membranes, placenta, trophoblasts, and decidua were compared with those from T47D breast cancer cells, using Western blotting techniques with the PR antibodies that detected the cytoplasmic isoform in the immunohistochemistry studies (Fig. 1 and Table 2), several PR-immunoreactive proteins were observed in T47D breast cancer cell extracts in line with previous studies (8, 19, 21), whereas the tissues at the fetal maternal interface revealed a major PR isoform with a relative molecular mass of approximately 60 kDa (Fig. 3). This isoform was confirmed as the PR-C isoform, as previously reported in human breast cancer cells (5, 23), human fetal membranes (19), and the guinea pig cervix (21), with recombinant protein and preadsorption studies (Fig. 3). Other progesterone target tissues, such as endometrium (data not shown) and myometrium, possessed the 116-kDa PR-B, 94-kDa PR-A, and 60-kDa PR-C isoforms but only with the C-20 antibody (Fig. 3).
Discussion
The role of PRs in human gestation and parturition is to maintain pregnancy. In most mammals, the stability of the relationship in the fetal-maternal interface is disturbed by the fall of progesterone production by the placenta, with the concomitant softening of the cervix, rupture of the fetal membranes, and initiation of highly synchronized high-pressure contractions of the myometrium that characterizes labor (24). In the human, similar events occur in the same coordinated manner except there is no loss of systemic progesterone (24). A hypothesis presented (17), that a switch in PR isoforms from the more transcriptionally dominant PR-B to the less active PR-A isoform must therefore occur in these tissues, has been convincingly supported by evidence in the myometrium (25, 26) and cervix (27, 28) and less convincingly in the amnion and maternal decidua (19).
In the present study, we have shown that the fetal membranes (amnion epithelial cell and chorionic cytotrophoblast) contain mainly high levels of PR-C (Fig. 1 and Table 2), whereas the placental cytotrophoblast is devoid of PR and the placental syncytiotrophoblast contains both PR-C and PR-A (Table 2). By contrast, the maternal decidua contains not only PR-A, as its major isoform, but also PR-B, PR-S, and the cytoplasmic PR-C. These data differ from those recently reported (19) in which PR-B and PR-A isoforms were observed in amnion nuclear extracts, yet paradoxically the antibody used failed to show immunoreactive PR by immunohistochemistry and theoretically should not detect the PR-C, PR-S, or PR-M isoforms in Western blotting because the epitope for this antibody is found only at the N terminal region of the PR-A isoform (29). In the present study, no PR-B or PR-A isoforms were detected in the amnion or chorion and the hypothesis that progesterone receptor switching occurs within these tissues at term or in labor (17) is not supported. Although we cannot rule out the presence of a small amount of PR-B or PR-A isoforms being present in the nuclei of amnion epithelial cells, despite a lack of expression of these isoforms at the mRNA level (Fig. 2), the present study indicates that PR-B and PR-A are of little importance in relation to PR-C in fetal membranes. Additionally, the PR immunoreactivity in the present study that we consider represents the C isoform was confined to the cytoplasm of the amniotic epithelium and chorionic cytotrophoblast and was the major isoform present, whereas no cytoplasmic immunoreactivity was detected in the aforementioned study (19).
These data are qualitatively similar to that obtained in the baboon (30), in which PR was not observed in the amnion or chorion, but strong nuclear and weak cytoplasmic staining was observed in the decidua with the JZB39 rat anti-PR monoclonal antibody. Observations obtained with JZB39 (31), which has been shown to detect only the PR-C isoform in myometrium and not T47D extracts by immunoblotting, were dependent on protein concentrations (31). These data suggest that protein loading in immunoblotting is critical for good PR-C detection. Indeed, PR-C was weakly represented on Western blotting in a previous study (19), whereas our data show intense PR-C expression (Fig. 3) for this exact reason. Alternatively, these discrepant observations may be related to the antibodies used; the use of nuclear extracts compared with whole cell extracts; or the presence of contaminating cytoplasm/tissues in the previous study (19).
In agreement with our studies, Goldman et al. (19) showed that PR-A is expressed in the nucleus of the decidua but absent in the amnion by immunohistochemistry but paradoxically found all isoforms present by Western blotting. By contrast, our Western blot studies indicate the major PR isoform in the fetal membranes (amnion and chorion) is C and that it is localized within the cytoplasm, without immunoreactivity within the nuclei of the same cells, and, therefore, we consider that the relevance of the changes in levels of the PR isoforms detected within the amniotic nuclear extracts of labor (19) should be considered with caution.
Using antibodies that detect all known PR isoforms, we demonstrated that the major isoform in the amnion, chorion, and syncytiotrophoblast is the 60-kDa PR-C isoform (Fig. 3). The absence of PR-B and PR-A transcripts in these tissues (Fig. 2) confirmed these findings. By contrast, the major isoform in the term decidua is PR-A, but PR-B, PR-S, and PR-C isoforms are also expressed. In light of the study by Goldman et al., these data need further clarification but suggest that PR-C is an important molecule in the fetal membranes, decidua, and syncytiotrophoblast. These data also suggest that a reevaluation of the roles of PR in the fetal-maternal interface is needed, and the idea that PR-A simply acts as a dominant-negative regulator of PR-B action is not applicable to these tissues (17).
The mechanism involved in the synthesis of the PR-C isoform is unknown, but limited evidence suggests that it may be produced using a third estrogen-dependent promoter with an AUG at amino acid 595 being the translational start site (5). The demonstration that PR-C isoforms in the human breast cancer cell do not arise from proteolysis of larger PR isoforms (9) and that specific PR-C mRNA transcripts are present in human fetal membranes and placenta (this study) strongly suggests that the PR-C isoform is generated through promoter-specific transcription. The function of PR-C is unclear, although it may act as modulator of PR-A and PR-B transcriptional activities in those cells that produce these isoforms (9). In the human amnion and chorion, dominant-negative regulation of PR-B and PR-A transcriptional control seems unlikely in the absence of any measurable PR-A or PR-B isoforms and points toward PR-C-specific functions. Because PR-C lacks a full DNA binding domain (9) but has a nuclear localization signal and two dimerization domains, the intriguing possibility that PR-C may associate with other transcriptional elements to modulate gene transcription is raised. However, the cytoplasmic location and relatively high levels of this protein are suggestive of a localized function in the cytoplasm rather than a nuclear genomic function and that this localization is important for sustained gestation. It now seems imperative that the regulation and role of the PR-C isoform is investigated to further our understanding of the role of progesterone in these tissues during human parturition.
Acknowledgments
The authors thank Reshma Bharkhada for excellent technical assistance and Dr. O. Habayeb and Mr. M. Habiba (University Hospitals Leicester) and Prof. L. Jones (St. Bartholemew’s Hospital, London, UK) for assistance in the procurement of clinical specimens and tissue extracts. The human PR-A and PR-C expression plasmids were kind gifts of Dr. Christine Clarke (Westmead Institute for Cancer Research, University of Sydney, Australia) and Dr. Kathryn Horwitz (University of Colorado Health Sciences Center, Denver, CO), respectively.
Footnotes
The authors have no conflicts of interest.
First Published Online October 27, 2005
Abbreviations: AMV, Avian myeloblastosis virus; PR, progesterone receptor; RT, reverse transcriptase.
Accepted for publication October 19, 2005.
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