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Department of Obstetrics and Gynecology (A.G.B., C.D.M., P.C.K.L.), University of British Columbia, Vancouver, British Columbia, Canada V6H 3V5; and Department of Obstetrics and Gynecology (C.-S.C.), Taipei Medical University, Taipei, Taiwan, Republic of China
Address all correspondence and requests for reprints to: Peter C. K. Leung, Ph.D., Department of Obstetrics and Gynecology, University of British Columbia, Room 2H-30, 4490 Oak Street, Vancouver, British Columbia, Canada V6H 3V5. E-mail:peleung{at}interchange.ubc.ca.
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Introduction |
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The formation and organization of the human placenta involves the active remodeling of the extracellular matrix of the maternal decidua (8, 9, 10, 11). Consequently, proteinases and their associated inhibitors are believed to play key roles in implantation and placentation. In particular, the spatiotemporal expression of two major classes of proteinases; urokinase plasminogen activator (uPA) and matrix metalloproteinases (MMPs) in both the maternal and fetal compartments have been shown to contribute to these two interrelated developmental processes. The secretion of plasminogen activator inhibitor-1 and tissue inhibitors of metalloproteinases by both the invading extravillous cytotrophoblasts and surrounding decidual cells counterbalance the proteolytic activities of uPA and MMPs at the maternal-fetal interface in an autocrine and/or paracrine manner.
We recently determined that GnRH I and the second mammalian form of this hormone, GnRH II, regulate the balance among MMP-2, MMP-9/tissue inhibitor of metalloproteinase-1, and uPA/plasminogen activator inhibitor-1 expression levels in primary cultures of extravillous cytotrophoblasts and decidual stromal cells isolated from first-trimester tissues (12, 13, 14, 15). Taken together, these observations suggest that GnRH I and GnRH II play key regulatory roles in both trophoblast invasion and the remodeling of the decidual extracellular matrix. Although GnRH I and GnRH II have been detected in the human placenta (16, 17, 18) and endometrial tissues throughout the menstrual cycle (19, 20), the expression pattern(s) of these two hormones among the distinct subpopulations of cells that constitute the human maternal-fetal interface, particularly during early pregnancy, remain poorly characterized. To address these outstanding issues, we performed a comprehensive survey of the cellular distribution of GnRH I and GnRH II mRNA and protein levels in human placental or decidual tissues and cells in vivo and in vitro. These studies not only provide useful insight into the spatiotemporal expression of GnRH I and GnRH II at the maternal-fetal interface but also identify model systems that can be used to study the regulation and function of these two hormones during human implantation and placentation.
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Materials and Methods |
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Tissue samples of first-trimester chorionic villi or decidua parietalis (nonimplantation site to avoid trophoblast contamination) were obtained from women undergoing elective termination of pregnancy. The gestational ages of these tissues ranged from 8 to 12 wk of pregnancy. Term placental tissues were obtained from women undergoing cesarean section. The use of these tissues was approved by the committee for ethical review of research involving human subjects, University of British Columbia. All patients provided informed written consent.
Samples of the placental or decidual tissue specimens were then snap frozen for later extraction of total RNA, fixed for immunohistochemical analysis, or processed for cell isolation and/or organ culture.
Cell purification and culture
To further localize GnRH I and GnRH II to the distinct subpopulations of cells that constitute the human placenta and decidua, primary cultures of extravillous cytotrophoblasts, villous cytotrophoblasts, or decidual stromal cells were purified using established protocols.
Extravillous cytotrophoblasts were propagated from first-trimester placental tissues (n = 3) as described by Graham et al. (21). The cellular characteristics of these primary cell cultures suggests that this method isolates the highly invasive subpopulation(s) of extravillous cytotrophoblasts that detach from extravillous cytotrophoblast columns and invade into the underlying maternal tissues during early pregnancy (8, 22, 23). Briefly, chorionic villi were washed thoroughly in DMEM (Life Technologies, Inc., Burlington, ON) supplemented with antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin). The villi were minced finely and plated in 25-cm2 tissue culture flasks containing DMEM supplemented with antibiotics and 10% heated-inactivated fetal bovine serum (FBS). The fragments of chorionic villi were allowed to adhere for 2–3 d, after which the nonadherent material was removed. The villous explants were cultured for a further 10–14 d with the culture medium being replaced every 48 h. Mononucleate extravillous cytotrophoblasts were separated from the chorionic villous explants by trypsin digestion and plated in 60-mm culture dishes containing the culture medium described above. The purity of the resultant extravillous cytotrophoblast cultures was determined by immunostaining for cytokeratin. Only cultures that were 100% immunostained for this epithelial cellular marker were used in these studies (data not shown).
Villous cytotrophoblasts were purified from human term placenta (n = 3) as previously described by Kliman et al. (4). This method, which uses serial trypsin-Dnase digestions in Ca2+- and Mg2+-free media followed by Percoll gradient centrifugation to purify cells, yields a highly enriched preparation of mononucleate villous cytotrophoblasts. After isolation, the cells were resuspended and plated in 3.5-cm culture dishes containing the culture medium described above. These mononucleate cells, which undergo spontaneous differentiation and fusion to form large multinucleated syncytium over time, were harvested after 12 or 48 h of culture.
Stromal cells were purified from decidual tissues (n = 3) by enzymatic digestion and mechanical dissociation using a protocol previously described (12). Briefly, pieces of the decidua were minced and subjected to enzymatic digestion with collagenase (type IV) and hyaluronidase (type I-S). The isolated stromal cells were then pelleted by centrifugation and plated in 60-mm culture dishes containing DMEM supplemented with antibiotics and 10% FBS. The purity of the decidual stromal cell cultures (passage 2) to be used in these studies was determined by immunocytochemical staining for vimentin, cytokeratin, muscle actin, and factor VIII (data not shown). These cellular markers have been used to determine the purity of human endometrial cell cultures (24). As defined by these criteria, only decidual stromal cell cultures that contained less than 1% epithelial or vascular cells were subsequently included.
Establishment of a chorionic villous-decidual tissue explant coculture system
Cocultures (n = 3) of chorionic villi and decidual tissue explants were established on a three-dimensional gel of rat tail tendon collagen as previously described (25, 26). This model system is believed to mimic the structural and cellular development of extravillous cytotrophoblast columns that form at the maternal-fetal interface and anchor the placenta to the uterine wall (5). The cocultures were established using 1- to 2-mm cubes of first-trimester decidual and chorionic villous tissues. The decidual tissue explants were first placed in 3.5-cm culture dishes containing 0.1 ml of a mixture of rat tail collagen (3 mg/ml) in acetic acid and 10 x DMEM (1:9 vol/vol) with the pH adjusted to neutral using a solution of sodium bicarbonate. Warming this mixture to 37 C caused it to gel after 15–20 min. After the gel had formed, a piece of chorionic villous, suspended in 0.5 ml DMEM, was placed on top of the exposed surface of the decidual tissue explant. The plates were then flooded with DMEM containing antibiotics and supplemented with 10% FBS after a further 2 h of culture. Control cultures of chorionic villi (n = 3) or decidua alone (n = 3) were similarly established at the surface of collagen gels (data not shown).
The cocultured or control tissues were maintained for a further 48 h before being embedded in Tissue-Tek OCT II compound and snap frozen in liquid nitrogen. Blocks of cocultured tissues were cut in full to obtain complete information on the three-dimensional aspect of the tissue contact sites. Cryostat sections (7 µm) were prepared, air dried, and stored at -20 C. The sections were warmed to room temperature and rinsed in Tris-buffered saline [0.05 M Tris-HCl (pH 7.6), 0.15 M NaCl] immediately before immunohistochemical analysis.
Isolation of total RNA and generation of first-strand cDNA
Total RNA was extracted from samples of first-trimester placenta (n = 3) or decidua (n = 3), term placenta (n = 3), or the primary cultures of trophoblasts or decidual stromal cells using the phenol-chloroform method of Chomczynski and Sacchi (27). To verify the integrity of the RNA, aliquots of the total RNA extracts were electrophoresed in a 1% (wt/vol) denaturing agarose gel containing 3.7% formaldehyde and the 28 S and 18 S rRNA subunits visualized by ethidium bromide staining. The purity and concentration of the total RNA present in each of the extracts were determined by optical densitometry (260/280 nm) using a Du-64 UV-spectrophotometer.
Aliquots (1 µg) of the total RNA extracts prepared from the placental or decidual tissues or cells were reverse transcribed into cDNA using a first-strand cDNA synthesis kit according to a protocol recommended by the manufacturer (Amersham Pharmacia Biotech, Oakville, Canada).
Semiquantitative PCR
Nucleotide sequences specific for human GnRH I or GnRH II (GenBank accession nos. X15215 and NM_178331, respectively) and that also spanned exons 1–4 of these two genes were identified using the BLAST (basic local alignment search tool) computer program (National Center for Biotechnology Information, Bethesda, MD). Forward and reverse oligonucleotide primers corresponding to these DNA sequences and primers specific for the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or human E-cadherin (E-cad), a marker of mononucleate villous cytotrophoblasts (28, 29), both of which served as internal controls for these studies, were synthesized at the NAPS Unit, University of British Columbia. The specific nucleotide sequences of these primers, the optimized PCR conditions for each of these primer sets, and the expected sizes of the PCR products are listed in Table 1
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. All PCRs were repeated on three independent occasions. To confirm the specificity of the primers, the resultant PCR products were subcloned into the PCR II vector by blunt-end ligation (Invitrogen, Carlsbad, CA) and subjected to nucleotide sequence analysis using an automated DNA sequence analyzer (Applied Biosystems, Foster City, CA) employing Taq DiDeoxy reagents (data not shown).
Immunohistochemical analysis
Immunohistochemistry was performed using sections prepared from permanent paraffin blocks containing first-trimester chorionic villi (n = 3), decidual tissues (n = 3), or the frozen sections prepared from the cocultured tissue explants. The tissue sections were immunostained using polyclonal antibodies directed against either human GnRH I or GnRH II (kind gift from Dr. V. A. Ferro, University of Strathclyde, Glasgow, UK) on three independent occasions. The specificity of these antibodies has been described in detail elsewhere (30). The slides were also stained with commercially available monoclonal antibodies directed against human cytokeratin filaments 8 and 18 (Becton Dickson, San Jose, CA) or human E-cad (Transduction Labs, Lexington, KY). Nonspecific isotype-matched antibodies were used as negative controls.
Sequential incubations were performed according to the methods of Cartun and Pedersen (31) and included 10% normal horse serum for 30 min, primary antibody at 37 C for 1 h, secondary biotinylated antibody at 37 C for 45 min, streptavidin-biotinylated horseradish peroxidase complex reagent at 37 C for 30 min, and three 5-min washes in PBS. The sections were then exposed to chromagen reaction solution (0.035% diaminobenzidine and 0.03% H2O2) for 10 min, washed in tap water for 5 min, counterstained in hematoxylin, dehydrated, cleared, and mounted.
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Results |
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GnRH I mRNA transcripts were readily detectable in the total RNA extracts prepared from first-trimester chorionic villi or decidual tissues (Fig. 1). GnRH II mRNA was also detected in these two tissues, albeit at lower levels than those encoding the classical form of this hormone. In contrast, GnRH I, but not GnRH II, mRNA transcripts were detected in total RNA extracts prepared from term placenta (Fig. 1).
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GnRH I expression was immunolocalized to the villous cytotrophoblasts and syncytial trophoblast of first-trimester chorionic villi (Fig. 2). In contrast, immunostaining for GnRH II was detected only in the mononucleate villous cytotrophoblast layers (E-cad positive) and not the multinucleated syncytial trophoblast (E-cad negative). Intense immunostaining for GnRH I and GnRH II was also observed in the columns of mononucleate extravillous cytotrophoblasts (E-cad positive) present in the first-trimester placental tissues (Fig. 3), whereas we failed to detect significant expression levels of either hormone in the mesenchymal core or blood vessels of these chorionic villi.
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). The expression of both forms of this hormone appeared to be localized primarily to the large decidual stromal cells of this endometrial cellular compartment.
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GnRH I and GnRH II mRNA transcripts were readily detectable in primary cultures of highly invasive extravillous cytotrophoblasts propagated from explants of first-trimester chorionic villi (Fig. 5). In agreement with our in vivo observations, GnRH I and GnRH II mRNA transcripts were readily detectable in stromal cells isolated from first-trimester decidual tissues (Fig. 5).
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). In contrast, GnRH II mRNA levels were high in the villous cytotrophoblasts cultured for 12 h but declined after 48 h, as the cells underwent terminal differentiation and fusion to form multinucleated syncytium, a developmental process associated with a decrease in E-cad mRNA levels. Localization of GnRH I and GnRH II in cocultured chorionic villi and decidua
Cytokeratin immunostaining of the cocultured tissue explants demonstrated that the integrity of the villous cytotrophoblast and syncytial trophoblast cell layers of the chorionic villi was maintained in areas not at the interface with the decidua (Fig. 6). However, in areas of contact between the two tissues, there was apparent cytotrophoblast proliferation, erosion of the syncytial trophoblast, and formation of extravillous cytotrophoblast columns that extended into the decidua.
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). Similarly, GnRH II was found to be expressed in the mononucleate villous and extravillous cytotrophoblasts but was not detected in the outer multinucleated syncytial trophoblast. GnRH I and GnRH II expression was also found to be maintained in the cultured decidual tissue explants. |
Discussion |
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In agreement with our observations, GnRH I has been detected in first-trimester and term chorionic villi with the levels of the mRNA transcript encoding this hormone remaining relatively constant in the human placenta throughout gestation (16, 32, 33). However, there have been conflicting results regarding the distribution and expression levels of GnRH I within the distinct trophoblast layers of these placental tissues. For example, GnRH I mRNA transcripts have been found to be present in villous cytotrophoblasts at equivalent or higher levels than the syncytial trophoblast of first-trimester placenta (32, 33), whereas GnRH mRNA was detected only in the outer trophoblast layer of term placentae (34). These differences may be attributed to the sensitivity of the in situ hybridization histochemical techniques employed in these studies and/or represent the depletion of villous cytotrophoblasts in the human placenta with gestational age (4, 7). In these studies, we demonstrated that GnRH I is expressed in the villous cytotrophoblast and syncytial trophoblast of first-trimester chorionic villi and that the levels of the mRNA transcript encoding the classical form of this hormone remain relatively constant during the terminal differentiation and fusion of villous cytotrophoblasts in vitro. In addition, we determined that GnRH I is expressed in the cytotrophoblasts at the tips of first-trimester chorionic villi that proliferate to form extravillous columns in vivo and in vitro and in primary cultures of highly invasive extravillous cytotrophoblasts propagated from these placental tissues. Taken together, these observations demonstrate that GnRH I is widely expressed among the distinct subpopulations of trophoblasts present at the maternal-fetal interface.
To date, GnRH II expression has only been detected in explants of first-trimester placental tissues in vitro (18). Here we demonstrate that there is a marked decrease in placental GnRH II mRNA levels with gestational age. This decrease in GnRH II mRNA levels may be attributed to the cellular distribution of this hormone within the placenta, being localized to the villous cytotrophoblasts and extravillous cytotrophoblast columns of first-trimester chorionic villi in vivo and in vitro and not the outer multinucleated syncytial trophoblast, the predominant trophoblast subtype present in this dynamic tissue at term (4, 7). GnRH II mRNA levels were also found to be high in primary cultures of villous and extravillous cytotrophoblast cultures. However, there was a marked decline in the levels of this mRNA transcript as the villous cytotrophoblasts underwent spontaneous terminal differentiation and fusion to form multinucleated syncytium in culture. Collectively, these observations demonstrate that unlike GnRH I, GnRH II expression is restricted to the mononucleate cytotrophoblasts of the human placenta.
In the human placenta, GnRH I regulates the secretion of human chorionic gonadotropin by the syncytial trophoblast in both an autocrine and paracrine manner (17, 35, 36). GnRH II has also been shown to be a potent regulator of human chorionic gonadotropin secretion by placental tissue explants and be less resistant to degradation by placental C-peptidases than its classical counterpart (18, 37). However, the restricted expression of GnRH II to the mononucleate cytotrophoblasts of the human placenta suggests that the regulatory effects of this hormone on the biochemical differentiation/function of the syncytial trophoblast is elicited in a paracrine manner.
Previous studies have demonstrated that both GnRH I and GnRH II are constitutively expressed in the glandular epithelium and stroma of the human endometrium throughout the menstrual cycle although maximum levels of these two hormones have been detected during the secretory phase (19, 20). In addition, GnRH I has been detected in the decidua of early pregnancy and endometrial stromal cells induced to undergo steroid-mediated decidualization in vitro (38, 39). Here we report that GnRH II is also expressed by first-trimester decidua and primary cultures of stromal cells isolated from these tissues. To date, the biological significance of endogenous GnRH I and GnRH II expression in the human endometrium remains poorly understood.
The biological actions of GnRH I and GnRH II on human tissues and cells are believed to be mediated by the specific binding of these two hormones to the GnRH I receptor (GnRHR) I (40). GnRHR I has been detected in the human placenta and endometrium and been further localized to villous cytotrophoblasts and syncytial trophoblast in vivo and in vitro (35), primary cultures of extravillous cytotrophoblasts propagated from first-trimester chorionic villi (41), and endometrial stromal cells (39). Furthermore, both low binding affinity/high-capacity binding and high binding affinity/low-capacity binding sites for GnRH I are present in the human endometrium (42) and GnRH II has been shown to bind the GnRHR present in the human placenta with higher affinity than GnRH I (18). Collectively, these observations suggest that the biological actions of these two hormones are elicited by distinct receptors present in these two tissues. Recently a gene encoding a second receptor for GnRH (GnRHR II) has been identified in the human genome (40, 43, 44). Although a full-length mRNA transcript encoding this second form of human GnRHR has not been isolated, GnRHR II mRNA has been detected in total RNA extracts prepared from human placenta and in normal and malignant endometrial tissues and cells (40, 45). To date, the cellular distribution of this mRNA transcript within these two tissues has not been determined. In view of the widespread, cellular distribution of GnRHR I and the presence of a putative GnRHR II in the human placenta and decidua, we cannot discount the possibility that our immunolocalization of GnRH I and GnRH II and in these two tissues may be owing to, at least in part, the detection of GnRH I and/or GnRH II bound to their mutual and/or distinct receptor(s). However, the presence of GnRH I and GnRH II mRNA transcripts in the primary cultures of trophoblasts and decidual stromal cells examined in these studies suggests that these distinct cell types have the potential to secrete one or both forms of this hormone.
In summary, we have determined that GnRH I is widely expressed among the distinct subpopulations of trophoblasts of the placenta, whereas the expression GnRH II is restricted to mononucleate villous and extravillous cytotrophoblasts. Both GnRH I and GnRH II were also detected in first-trimester decidua and found to be coexpressed in primary cultures of decidual stromal cells. Collectively these observations suggest that both of these hormones are capable of eliciting their biological actions within and between these two dynamic reproductive tissues in an autocrine and paracrine manner. Furthermore, these studies identify model systems that can be used to study the regulation and function of GnRH I and GnRH II during human implantation and placentation.
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Acknowledgments |
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Footnotes |
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This work was supported by an operating grant from the Canadian Institutes of Health Research (to P.C.K.L. and C.D.M.). P.C.K.L. is the recipient of a Senior Investigatorship from the Michael Smith Foundation for Health Research. C.D.M. is a Career Investigator of the British Columbia Research Institute for Children’s and Women’s Health.
Abbreviations: E-cad, E-cadherin; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GnRHR, GnRH I receptor; MMP, matrix metalloproteinase; uPA, urokinase plasminogen activator.
Received September 18, 2003.
Accepted December 16, 2003.
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