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Research Article |
Department of Obstetrics and Gynecology,4 University of Illinois at Chicago, Chicago, Illinois 60212-7313
Department of Obstetrics and Gynecology,5 University of Missouri, Columbia, Missouri 65212
ABSTRACT
Chorionic gonadotropin (CG) plays an important role in establishing a receptive endometrium by directly modulating the function of both endometrial stromal and epithelial cells in the baboon. The focus of this study was to characterize changes in CG receptor (LHCGR, also known as CG-R) expression during the menstrual cycle and early pregnancy, particularly during decidualization. LHCGR was localized by using a peptide-specific antibody generated against the extracellular domain. Immunostaining was absent in any of the cell types during the proliferative phase of the cycle. In contrast, during the secretory phase, both luminal and glandular epithelial cells stained positively. Stromal staining was confined to the cells around spiral arteries (SAs) and in the basalis layer. This stromal staining pattern persisted at the implantation site between Days 18 and 25 of pregnancy and after CG infusion. However, as pregnancy progressed (Days 40 to 60), staining for LHCGR was dramatically decreased in the stromal cells. These data were confirmed by nonisotopic in situ hybridization. To confirm whether the loss of LHCGR was associated with a decidual response, stromal fibroblasts were decidualized in vitro, and cell lysates obtained after 3, 6, and 12 days of culture were analyzed by Western blotting. LHCGR protein decreased with the onset of decidualization in vitro, confirming the in vivo results. Addition of CG to decidualized cells resulted in the reinduction of LHCGR in the absence of dbcAMP. We propose that CG acting via its R on stromal cells modulates SA in preparation for pregnancy and trophoblast invasion. As pregnancy progresses, further modification of SA by migrating endovascular trophoblasts and subsequent decidualization results in the downregulation of LHCGR. This inhibition of LHCGR expression also coincides with the decrease of measurable CG in peripheral circulation.
decidua, female reproductive tract, human chorionic gonadotropin, implantation, pregnancy
INTRODUCTION
Chorionic gonadotropin (CG), one of the earliest embryonic signals secreted by the primate blastocyst, is a glycoprotein hormone belonging to the same family as follicle-stimulating hormone (FSH), luteinizing hormone (LH), and thyroid-stimulating hormone (TSH). During implantation and pregnancy, CG is primarily secreted by the syncytiotrophoblast. The profile in the peripheral circulation during pregnancy in the baboon shows an increase between Day 12 and Day 15, reaching a peak at Day 27 of pregnancy and declining to nonpregnant levels by Day 50 [1]. Our studies have demonstrated that in addition to providing luteotrophic support, CG also modulates the endometrium in preparation for implantation [2]. Infusion of CG into the uterine cavity of cycling baboons, in a manner that mimics normal blastocyst transit, induces a plaque response in the luminal epithelium, an induction of 
-smooth muscle actin (ACTA2, also known as SMA) in the stromal fibroblasts, and an increase of glycodelin expression and secretion in the glandular epithelium [2].
Successful establishment of pregnancy depends not only on the embryo quality but also on the transformation of uterine stromal fibroblasts into fully differentiated decidual cells. During the process of decidualization, stromal cells change morphologically and biochemically into polygonal, secretory cells and begin to express specific decidual proteins such as prolactin (PRL) and insulin-like growth factor binding protein 1 (IGFBP1). Although the biochemical characteristics of decidual cells have been extensively studied, many cellular and molecular events associated with the transformation of stromal fibroblasts remain to be elucidated. As previously reported [3], CG may stimulate differentiation of stromal uterine fibroblasts into decidual cells. It was also observed that CG plays an important role in the control of endometrial vascularization [4, 5] and promotes synthesis of nitric oxide (NO) [6], an important factor facilitating angiogenesis and vasodilation in the endometrium. In addition, numerous studies have demonstrated decidualization of primate endometrial stromal cells in vitro in the presence of hormones and growth factors [7, 8]. In our in vitro baboon (Papio anubis) and human models, we have demonstrated that decidual transformation of stromal cells occurs in the presence of hormones and embryonic factors such as interleukin 1 beta (IL1B, also known as IL-1ß) or cAMP analogue, dbcAMP [9, 10]. These data were further substantiated with in vivo studies in which we demonstrated that infusion of CG together with IL1B into the uterine lumen initiates a decidual response similar to that observed in early pregnancy [11].
The CG/LH receptor (LHCGR) is a member of the subfamily of glycoprotein hormone receptors within the superfamily of G protein–coupled receptors (GPCRs). The presence of extragonadal LHCGR has been reported in many reproductive [12] and nonreproductive [13, 14] tissues in the human. Since 1990, LHCGR has been known to be present in human endometrium [8]. Although its distribution was extensively studied, still many unanswered questions remain regarding its role and regulation during the menstrual cycle, implantation, and pregnancy. Our previous studies have shown that CG directly modulates the function of endometrial epithelial cells via the MAPK pathway [15], which is different from the conventional signal-transduction pathway induced by CG in gonadal tissues [16]. Targeted deletion of the LHCGR receptor results in an altered endometrial phenotype and disrupted gene expression [17]. In addition, the LHCGR-null animals do not demonstrate a complete decidualization response and have lower implantation rates when wild-type donor embryos are transferred into the uterus [18].
The purpose of the current study was to investigate the distribution and regulation of endometrial LHCGR during the menstrual cycle and early pregnancy, particularly during the decidualization process. Studies were performed by using endometrial tissue from cycling baboons as well as baboon stromal fibroblasts and human uterine fibroblasts (HuF) cells in vitro.
MATERIALS AND METHODS
Materials
Chorionic gonadotropin receptor (LHCGR) antibody was made in male rabbits against a synthetic peptide, corresponding to amino acids 257–271 of the extracellular domain of the human LH/CG receptor (Exon 9: aa 257–271 Gene Bank Accession S57793), conjugated to keyhole lymphocyte hemaglutinin. The monoclonal antibody to IGFBP1 (B2H10) was kindly provided by Dr. Stephen C. Bell (University of Leicester, Leicester, United Kingdom). Recombinant human IL1B was obtained from R&D Systems Inc. (Minneapolis, MN). N6,2'-O-dibutyryladenosine 3:5'-cyclic monophosphate (dbcAMP), trichostatin A (TSA), and antibody against ACTB (ß-actin) were obtained from Sigma (St. Louis, MO). All cell-culture supplies and reagents of cell-culture grade were obtained from Life Technologies, Inc. (Gaithersburg, MD), Corning, Inc. (Corning, NY), Falcon (Franklin Lakes, NJ), Fisher Scientific (Itasca, IL), Sigma (St. Louis, MO), or Roche Molecular Biochemicals (Indianapolis, IN).
Tissue Collection
Endometrial baboon tissues (n = three to four/stage) were obtained from normally cycling, CG-treated, and pregnant animals housed at the Biological Resources Laboratory of the University of Illinois at Chicago. The day of ovulation was calculated as 2 days after the estradiol surge and was confirmed by prospective measurement of peripheral serum levels of estradiol [2, 19]. Tissue was obtained either at hysterectomy or after an endometriectomy. All experimental procedures were approved by the Animal Care Committee of the University of Illinois at Chicago.
LHCGR Immunohistochemical Detection
Baboon endometrial tissues were immersion-fixed in formalin solution for 24 to 48 h at room temperature, dehydrated in graded ethanol, cleared in xylene, and embedded in paraffin. Tissue sections were stained with polyclonal antibody generated against the extracellular domain of LHCGR (Exon 9: aa257–271: GenBank Accession S57793) at a 1:250 dilution and monoclonal IGFBP1 antibody at a 1:1000 dilution, as previously described [20]. The immunoreactive product was visualized by using an ABC Vectastain Kit (Vector Laboratories). Controls consisted of preimmune serum at the same dilution.
In Situ Hybridization
Baboon endometrial tissues were immersion-fixed in formalin solution for 24 to 48 h at room temperature, dehydrated in graded ethanol, cleared in xylene, and embedded in paraffin. Tissues were cut in 5-µm sections in RNAase-free conditions. The tissues were deparaffinized with xylene and rehydrated with graded ethanol into RNAase-free water. The tissues were hybridized with specific HPLC-purified, biotinylated 45mer oligonucleotide antisense and sense probes generated against exon 9 in the extracellular domain of LHCGR (GenBank Accession No. S57793), corresponding to the protein sequence against which the LHCGR antibody was generated. The Tm for the probes was 69.5°C. The tissues were pretreated with 50 mM Tris-HCl, pH 7.6, at 95°C for 20 min, cooled to room temperature for 20 min, and incubated with proteinase K (5 µg/ml) for 5 min. After extensive washing, endogenous peroxidase was quenched with 0.3% hydrogen peroxide in methanol for 20 min. Hybridization was conducted with the final concentration of the probes at 143 ng/µl at 45°C for 18 h. Subsequent stringent washes were performed at 55°C. The DAKOGenPoint catalyzed signal-amplification system for in situ hybridization (Dako, Carpinteria, CA) was used to detect the bound biotinylated probes by using a series of primary streptavidin-HRP, biotinyl-tyramide, and secondary streptavidin-HRP with alternating TBST washes. The chromagen diaminobenzidine, which is oxidized by the peroxidase enzymes, produced a dark brown precipitate at the site of hybridization. Tissues were counterstained with Harris hematoxylin. Controls included human placental tissue from 3 months of gestation (positive), and negative control included omission of the probes as well as absence of staining with the sense probes.
Isolation and Culture of Stromal Cells
Baboon stromal fibroblasts were isolated from endometrial tissues obtained after endometrectomy or hysterectomy [15]. Human uterine fibroblasts (HuF) cells were isolated from decidua parietalis dissected from the placental membranes after normal vaginal delivery at term [21, 22]. These studies were approved by the Institutional Review Board of the University of Illinois. In brief, scraped cells were digested in 0.1% collagenase, 0.02% deoxynuclease in calcium- and magnesium-free Hanks Balanced Salt Solution. Cells were isolated in the same manner as described previously [15, 16]. Cells were grown in 37°C, 5% CO2, trypsinized, and used for the experiments in passage number 3 or 4. Cell cultures were performed in phenol red–free RPMI-1640 medium supplemented with 0.1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum (FBS) depleted of steroids by treatment with dextran-coated charcoal (S-FBS). Cell purity was assessed with immunocytochemistry by using antibodies against cytokeratin (Dako Corp, Carpinteria, CA), vimentin (Zymed Laboratories, San Francisco, CA), and factor VIII (Dako). The purity of the stromal cell preparations used in these studies was greater than 95%.
Cell Culture Treatment and Immunodetection of LHCGR
Experiments were performed when cells reached 80% to 90% confluence. Culture medium with the appropriate treatment paradigms containing 2% heat-treated S-FBS was changed every 2 days. Cells were treated with 10 ng/ml IL1B or 100 µM dbcAMP, in the presence of 36 nM estradiol-17ß and 1 µM medroxyprogesterone acetate for 3, 6, and 12 days, in duplicate with the appropriate controls [10]. Culture supernatants were frozen, and cells were rinsed twice with ice-cold PBS and lysed on ice with RIPA lysis buffer. Twenty micrograms of proteins from cell lysates were separated on as SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked overnight in PBS containing 5% nonfat milk and 0.1% Tween-20, and then incubated with primary antibody for LHCGR at a 1:20000 dilution for 3 h followed by secondary goat anti-rabbit antibody labeled with horseradish peroxidase at a 1:3000 dilution for 1 h. Nonspecific IgG was used as procedural control. Immunocomplexes were visualized by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech). All membranes were reprobed with anti-ACTB antibody (Sigma), which served as a loading control. Densitometric analysis was performed with the NIH Scion Image Program. Results were expressed as a ratio of LHCGR to ACTB and presented as mean ± SEM.
To correlate in vitro and in vivo results, decidual tissues were obtained from baboons at Days 17, 25, 50, 60, 115, and 150 of pregnancy and at term. Total protein was extracted from 1 g of tissue by homogenization in 3 ml of RIPA lysis buffer containing protease inhibitors. Forty micrograms of proteins was separated on an SDS-PAGE and analyzed by Western blot as described earlier.
Chinese Hamster Ovary Cell Line
Chinese hamster ovary (CHO) cells (a gift from Karen Colley, Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago) express the LHCGR, which has a molecular weight similar to that identified in human uterine fibroblasts. The CHO cells do not undergo decidualization or differentiate when cultured under the same conditions as baboon or human stromal fibroblasts. Cells were cultured in Minimum Essential Medium alpha plus (Gibco), 0.1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% S-FBS until they reached 80% to 90% confluence. Cells were then treated under the same conditions required to induce decidualization in uterine stromal fibroblasts, as described earlier.
IGFBP1 Measurement
IGFBP1, as a marker of decidualization, was measured in culture supernatants. HuF and CHO cell-culture supernatants were collected and frozen at each time point during the experimental time course. IGFBP1 levels were measured by using an enzyme-linked immunosorbent assay (ELISA) kit (Diagnostic Systems Laboratories, Inc. Webster, TX) according to the manufacturer's specifications [10]
Statistical Analysis
Where appropriate, statistical analysis was performed with one ANOVA by using the GraphPad Instat Computer Program (San Diego, CA). Each of the experiments analyzed was repeated 3 times in duplicate.
RESULTS
LHCGR Localization During the Menstrual Cycle and Pregnancy
During the proliferative phase (Days 7 to 12 after menses), staining was faint in both stromal and epithelial cells (Fig. 1, A and B). In contrast, during the secretory phase (days 10–14 after ovulation, PO), the apical tips of both the luminal and glandular epithelial cells stained positively (Fig. 1C). Stromal staining during this phase was confined to the cells around spiral arteries (SAs) and the basalis layer (Fig. 1D). Treatment with CG downregulated LHCGR expression in the epithelial cells (Fig. 1E), whereas the staining in stromal cells around the SA remained intense (Fig. 1F). The presence of the LHCGR in luteal cells from a secretory phase corpus luteum was used as a positive control (Insert a; Fig. 1A).
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Similar to that in CG-treated animals, which mimics the early events of pregnancy (2), the presence of LHCGR in glandular epithelial cells was reduced during early pregnancy compared with the secretory phase. During early pregnancy (Days 18–30), positive stromal staining for LHCGR was strongest at the implantation site and in the cells surrounding SA (Fig. 2, A and B). However, as pregnancy progressed (Days 40–60), stromal staining of LHCGR was dramatically decreased in both the implantation site and around the SA (Fig. 2, C and D). The decrease in LHCGR localization was associated with the decidual response. This was confirmed by immunohistochemistry on adjacent sections with IGFBP1 staining (Fig. 2, E and F). The increase in IGFBP1 staining at the implantation site (Fig. 2F) is correlated with the decrease in the staining intensity for the LHCGR (Fig. 2C). IGFBP1 is a well-established marker of the decidualization in the primate [23].
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A similar staining pattern was observed when LHCGR mRNA expression was analyzed by in situ hybridization on baboon endometrial tissues during the menstrual cycle and pregnancy (Fig. 3). During the proliferative phase (Days 7–12 after menses), hybridization was very low or undetectable in both stromal and epithelial cells (Fig. 3A); however, during the secretory phase (Days 10–14 PO), hybridization was confined to the epithelial cells (Fig. 3B). After treatment with CG or during early pregnancy (Days 18–30), hybridization of LHCGR was most dramatic around the SA (Fig. 3, C and D). By Day 40 of pregnancy, LHCGR mRNA expression was decreased in the decidual cells, but continued to be expressed in the syncytiotrophoblast cells within the placental chorionic villi (Fig. 3E). The sense control is shown in Figure 3F.
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To confirm the immunohistochemistry and in situ hybridization data, protein extracts from decidual tissues at days 17, 25, 50, 60, 115, and 150 of pregnancy and at term were analyzed with Western blot. Decidual extracts from early pregnant animals (Days 17 and 25) confirmed immunocytochemical data. The polyclonal antibody cross-reacted with a protein band of approximately 80 kDa (Fig. 4). The molecular weight of the decidual LHCGR is similar to that reported in porcine ovarian follicular membranes [24] and endometrial cells [25], human endometrial stromal cells [26], human endometrial epithelial cell line (HES) [15], pig granulosa cells, and baboon corpus luteum (data not shown). After the onset of decidualization, at mid and late pregnancy, the LHCGR was undetectable in decidual extracts obtained from Day 50 of pregnancy onward (Fig. 4).
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Downregulation of LHCGR During In Vitro Decidualization
In vitro studies were conducted to investigate whether the observed decrease in LHCGR expression in vivo is associated with the decidualization process. Baboon stromal cells and HuF cells undergo decidualization in vitro when treated with IL1B or dbcAMP in the presence of steroid hormones. The changes in LHCGR were analyzed with Western blot. LHCGR was markedly downregulated in decidualizing baboon stromal cells after 12 days of treatment, particularly with dbcAMP (Fig. 5). The changes in presence of LHCGR protein observed in baboon stromal cells were further confirmed with human stromal fibroblasts. Treatment with hormones and dbcAMP for 3 days also caused a decrease in the LHCGR protein in HuF cells (Fig. 6). After 6 and 12 days of treatment, the LHCGR was almost undetectable both in dbcAMP- and IL1B-treated cells (Fig. 6).
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To confirm that the downregulation of the LHCGR was directly associated with the decidualization process, IGFBP1, a well-known marker of decidualization, was measured in the conditioned media. High levels of secreted IGFBP1 were detected after 3, 6, and 12 days of treatment with dbcAMP and hormones (Fig. 7, A-C). IL1B, in the presence of hormones, significantly increased IGFBP1 after 12 days of treatment (Fig. 7C). The increase in IGFBP1 secretion corresponded to the decrease in LHCGR protein shown in Figure 6.
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Sakai et al. [27] demonstrated that histone deacetylation is associated with the differentiation of human endometrial stromal cells into decidual cells. Further to confirm that the downregulation of LHCGR is associated with decidualization, HuF cells were treated with trichostatin A (TSA), an inhibitor of histone deacetylases (HDACs). LHCGR protein was analyzed with Western blot (Fig. 8). Treatment with TSA also caused a decrease in the LHCGR protein, demonstrating that other mechanisms that also induce stromal cell differentiation into decidual cells by transcriptional regulation of IGFBP1 result in the downregulation of LHCGR expression.
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CG and cAMP analogue treatments downregulate LHCGR transcripts in gonadal cells both in vivo and in vitro [28]. Our data would suggest a similar response to dbcAMP in uterine stromal fibroblasts (Figs. 5 and 6). In an attempt to elucidate the molecular mechanism of the LHCGR downregulation during decidualization, HuF cells were treated with CG in the presence or absence of dbcAMP, after decidualization. As shown in Figure 9, in the continued presence of dbcAMP, CG is unable to upregulate LHCGR protein in decidual cells. However, if dbcAMP was removed from the culture medium and the cells were treated with CG and hormones, the LHCGR was reexpressed within 24 h (Fig. 9). The ability of CG to regulate its own receptor was further confirmed by incubating undifferentiated stromal fibroblasts with CG. Treatment with CG resulted in a dose-dependent increase in LHCGR protein (Fig. 10). These data further substantiate stromal fibroblast response to CG in vivo during early pregnancy (Figs. 1 and 2).
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Regulation of LHCGR in CHO Cells
To investigate whether dbcAMP or IL1B treatment would induce the same changes in LHCGR expression in cells that do not undergo decidualization, parallel studies were performed on CHO cells. Cells were treated with the same concentrations of dbcAMP or IL1B in the presence of steroids for 3, 6, and 12 days, as described earlier for baboon endometrial stromal cells and HuF cells. No detectable changes in the expression of LHCGR were observed in any of the treatments at the different time points. Data from the 12-day treatment are shown in Figure 11. As expected, none of the treatments induced IGFBP1 (data not shown). Because cells of ovarian origin do not undergo decidualization, these observations suggest that LHCGR downregulation observed on endometrial stromal cells is related to the decidualization process.
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DISCUSSION
One of the most important requirements for the mammalian embryo to establish a successful pregnancy is its ability to implant in the mother's uterus. This process implies an appropriate spatiotemporal synchronic series of events in which the trophoblast cells of the embryo establish contact with the uterus and are able to attach to and intrude into the luminal epithelium. To accomplish this synchronous dialogue, the endometrium has to become receptive to signals derived from the embryo. When pregnancy is established, the endometrial stromal compartment forms the decidua, a morphologically and functionally gestation-specific tissue that represents the maternal side of the fetomaternal interface.
The human uterus expresses LHCGR [12], and treatment of women with hCG markedly alters endometrial morphology [29] and also inhibits apoptosis during the secretory phase [30]. We previously demonstrated that CG plays a key role during early pregnancy in the baboon by inducing changes in all three major cell types (luminal and glandular epithelium and stromal cells) within the uterine endometrium [2]. Different paracrine functions, including prostaglandin biosynthesis modulation [31, 32], glycodelin synthesis and secretion [33, 34], cytokine secretion [4], and the modulation of decidualization [4], have been proposed for CG in the endometrium. However, the expression of the LHCGR in the endometrium during the window of receptivity and during the establishment of pregnancy has yet to be resolved. In this context, the aim of this study was to analyze the expression of the LHCGR during the menstrual cycle and early pregnancy in the baboon endometrium.
Our studies demonstrate a cycle-dependent expression of LHCGR in the baboon endometrium. These studies confirm previous observations in the human endometrium [12, 35] and suggest the importance of CG signaling during the window of implantation. The LHCGR protein and mRNA expression pattern was completely modified in response to CG and after the establishment and progression of pregnancy. This decrease in LHCGR in decidual cells coincides with the period of pregnancy when measurable CG declines in the peripheral serum of pregnant baboons [1].
The specific expression of LHCGR in the stromal cells at the implantation site in early pregnancy is also correlated with the expression of ACTA2 (SMA) [2, 11] and cycloxygenase-2 (PTGS2, also known as COX-2) [36]. The induction of ACTA2 in these stromal cells is directly regulated by CG [2] and appears to be a necessary prerequisite to inhibit stromal cell apoptosis [30, 37] and enable decidualization to proceed [38, 39]. In addition, the coexpression of both LHCGR and PTGS2 in stromal cells at the implantation site during early pregnancy is in agreement with previous in vitro studies that demonstrated a direct effect of CG on PTGS2 gene expression and the induction of decidualization in human stromal cells cultured in vitro [32]. Thus the induction of the LHCGR in stromal cells at the maternal-fetal interface may play an essential role in inducing changes that are critical for both the establishment of pregnancy and the initiation of decidualization in the primate. Indirect evidence for the importance of the LHCGR in endometrial function can be gleamed from the LH receptor–null mouse. These animals have an abnormal uterine phenotype that cannot be corrected by estradiol and progesterone treatment alone and are unable to be implanted with wild-type donor blastocysts [18, 40].
In addition to the induction of LHCGR in stromal cells at the implantation site, the most dramatic increase in staining for the LHCGR was evident around the spiral arteries. Endometrial biopsies performed in women after CG treatment also demonstrated an increase in the presence of spiral arteries within the stroma [29]. Subsequent studies demonstrated that CG had a direct angiogenic effect on uterine endometrial cells [5]. The observed increase in LHCGR positive staining around the spiral arteries in early pregnancy was also correlated with positive staining for both nitric oxide isoforms (iNOS and eNOS) [6], supporting the hypothesis that CG may play a key regulatory role in angiogenesis and vascular development in the female reproductive tract [41]. Angiogenesis plays an important role in pregnancy-associated changes in the reproductive tract, and disturbances in uterine blood flow are associated with higher perinatal morbidity and mobility. Based on the potential importance of CG in ensuring an adequate blood flow, we suggest that this increase in LHCGR expression around the spiral arteries in early pregnancy is a maternal adaptation to ensure an adequate blood supply to the developing embryo.
In contrast to the dramatic increase in LHCGR-expressing stromal fibroblasts in early pregnancy, the onset of decidualization resulted in a profound downregulation of LHCGR expression both in vivo and in vitro. This downregulation appears to be a specific response in uterine stromal fibroblasts undergoing decidualization because CHO cells (which express the LHCGR) neither downregulated LHCGR nor produced IGFBP1 in response to the decidualizing stimuli in vitro. Licht et al. [42] also demonstrated that at the time of implantation, the human endometrium expresses the full-length LHCGR, but with the onset of decidualization, the expression pattern switched to truncated isoforms as a result of alternate splicing. It is unclear what the physiologic implications of this dramatic downregulation are or if this decrease in LHCGR expression is a direct effect of decidualization or if it occurs in response to the modification of the cellular phenotype.
Our studies suggest that cell transformation via cAMP or inhibition of HDACs is required for the downregulation of the LHCGR. Histone deacetylation catalyzed by HDACs promotes chromatin alterations for turning off genes and maintaining some genes in a repressed state. Sakai et al. [27] suggested that histone acetylation is involved in human endometrial stromal cells differentiation to decidual cells. Based on the effects we observed with TSA treatment, we suggest that chromatin remodeling may also affect the regulation of the LHCGR and that this occurs very rapidly. However, this does not appear to be an irreversible process because the removal from dbcAMP from the media in the continued presence of CG results in the repeated expression of LHCGR in the decidualized cells within 24 h. The mechanism by which this occurs is unclear, but it is possible that the level of protein expression may be regulated at the posttranscriptional level by cAMP [28, 43] because decidualization in vitro occurs as a consequence of increased endogenous cAMP in the presence of both estrogen and progesterone [9, 10]. In the rat ovary, binding of LH or CG to the receptor leads to an elevation of cAMP, which induces the expression of an LHCGR mRNA binding protein resulting in an increase in mRNA degradation and subsequent downregulation of LHCGR expression [43–45].
In this study, we showed that dbcAMP inhibits LHCGR protein expression during decidualization. Once decidualization is established, CG is able to upregulate the LHCGR in the absence of dbcAMP, but if dbcAMP is present, LHCGR expression is inhibited. This apparent contradiction with the downregulatory effects of CG on LHCGR expression found in rat ovary might be because CG does not induce cAMP production in primate endometrial cells [15]. It is conceivable to suggest that as a result of decidualization, which requires an increase in cAMP [9], uterine stromal cells may also upregulate the expression of a molecule such as LH receptor–binding protein to inhibit the expression of the LHCGR on these cells. The major decrease observed both at the mRNA and protein level in vivo could be explained by an increase in mRNA degradation, as reported in the rat ovary [28, 45]. Studies to elucidate the possible mechanisms by which CG upregulates its own receptor and cAMP downregulates LHCGR in endometrial stromal cells are currently under way in our laboratory.
In summary, we have characterized the LHCGR expression pattern during the menstrual cycle and in early pregnancy. We have further demonstrated that the LHCGR is markedly downregulated as a consequence of decidualization. These findings manifest the importance of CG during the secretory phase of the cycle and in early gestation, when the secretion of this hormone is high. We suggest that direct effects of CG on the endometrium via its receptor are necessary to induce the expression of specific genes that are important in modulating the decidualization process, the immune system, cell survival, and the vascularization at the maternal-fetal interface. The dynamic and cell-specific changes in LHCGR expression during the menstrual cycle and the establishment of pregnancy substantiate our previous studies [2, 15, 33, 46] in that CG, as an early embryonic signal, plays a critical role in establishing a receptive endometrium that is conducive to maintaining pregnancy in the primate.
ACKNOWLEDGMENTS
M. Szmidt is a graduate student in Department of Histology and Embryology, Warsaw Agricultural University, Poland. These studies were conducted at the University of Illinois in partial fulfillment of the requirements for the PhD degree from Warsaw Agricultural University.
FOOTNOTES
2 Correspondence: Asgi T. Fazleabas, Department of Obstetrics and Gynecology, University of Illinois at Chicago, 820 S Wood Street, M/C 808, Chicago, IL 60612. FAX: 312 996 8329; asgi{at}uic.edu 
3 These authors contributed equally to this work.
1 Supported by National Institute of Health (NIH) grant HD-42280 (to A.T.F.) and an Americas Fellowship supported by NICHD, NIH through D43TW00671 (to P.C.).
Received: 16 February 2006.
First decision: 11 March 2006.
Accepted: 12 July 2006.
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smooth muscle actin expression in the baboon (Papio anubis) uterus is associated with glandular secretory activity and stromal cell transformation. Biol Reprod 1995 53:596-606This article has been cited by other articles:
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