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a Departments of Obstetrics and Gynecology and
b Physiology and Biophysics, University of Illinois, Chicago, Illinois 60612
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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SMA) expression, an early marker for decidualization in the baboon in vivo, was induced spontaneously under normal culture conditions. Furthermore, a decrease in SMA expression was observed in cells producing high levels of IGFBP-1. Human cells produced significant levels of IGFBP-1 (p 
0.01) in response to short-term dbcAMP treatment (48 h) after 2 and 12 days of hormone treatment. However, baboon stromal cells required 17 days of hormonal treatment before cells became responsive to short-term dbcAMP treatment (p 0.01). Finally, human endometrial stromal cells expressed the protein kinase A regulatory subunits RI, RIß, RII, and RIIß whereas baboon stromal cells expressed RI, RII, and RIIß. No difference in the mRNA expression of these isoforms was observed in decidualized or nondecidualized cells of either human or baboon endometrium. Our observations indicate that baboon stromal cells can be induced to decidualize in vitro and that this requires dbcAMP in addition to hormones. This is the first report demonstrating in vitro decidualization in a nonhuman primate.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Insulin-like growth factor-binding protein-1 (IGFBP-1) is the major secretory product of the human [1] and baboon [2] decidualized endometrium and thus can be used as a biochemical marker of decidualization. In the human, decidualization begins at the end of each menstrual cycle and progresses if there is a pregnancy [3]. Numerous studies have demonstrated decidualization of human endometrial stromal cells in vitro in the presence of hormones and growth factors [4–6]. In the baboon, decidualization occurs only during pregnancy and requires the presence of a conceptus or a conceptus product [7]. Decidualization cannot be induced in vivo with long-term estrogen and progesterone treatment alone [8].
Baboon endometrial stromal cells demonstrate specific immunohistochemical staining for alpha smooth muscle actin (SMA) during early pregnancy. Alpha SMA staining eventually disappears at Days 32–40 of pregnancy [9]. This is coincident with the time these cells produce IGFBP-1, particularly at the implantation site [7]. In a simulated-pregnant baboon, hCG, estrogen, and progesterone were sufficient to induce SMA expression in stromal fibroblasts, but IGFBP-1 expression was not evident [8, 9]. These in vivo studies suggest that the conceptus regulates the decidualization process in the baboon.
The objective of this study was to define appropriate in vitro culture conditions to mimic changes associated with stromal cell decidualization in vivo in a nonhuman primate model. Specifically, SMA and IGFBP-1 were measured as markers for decidualizing stromal cells after treatment with estradiol-17ß, medroxyprogesterone acetate (MPA), and relaxin in the presence or absence of the cAMP analogue, N6,2'-O-dibutyryl cAMP (dbcAMP). Furthermore, a comparison of the regulatory transcripts of the protein kinase A (PKA) subunits—RI, RIß, RII, and RIIß—between decidualized and nondecidualized cells was done in order to investigate a potential mechanism of cAMP action during the decidualization process. Parallel studies with human endometrial stromal cells were done strictly for comparative analysis. Our studies suggest that while hormones alone are sufficient to decidualize human stromal cells in vitro, dbcAMP in conjunction with hormones is required for induction of this transformation in baboon stromal fibroblasts.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All cell culture supplies were obtained from Gibco BRL (Gaithersburg MD). Other reagents of cell culture grade were purchased from Fisher Scientific (Itasca, IL), Sigma Chemical Company (St. Louis, MO), or Boehringer-Mannheim (Indianapolis, IN).
Immunocytochemistry
Endometrial tissues were obtained from cycling (Day 9-10 postovulation), hCG- and steroid-treated (simulated-pregnant), and pregnant baboons. The day of ovulation was designated to be 2 days after the estradiol surge and was confirmed by prospective measurement of peripheral serum levels of estradiol [8]. In order to simulate pregnancy, normally cycling animals received increasing dosages of hCG (Profasi; Serono Laboratories, Inc., Norwell, MA) in addition to estradiol and progesterone via s.c. Silastic implants (Dow-Corning, Midland, MI). This treatment regimen resulted in peripheral levels of hormones comparable to those of pregnancy [8]. The binding of antibodies to SMA and IGFBP-1 on paraffin-embedded tissue was visualized using an ABC Vectastain kit (Vector Laboratories, Inc., Burlingame, CA) as previously described [7, 9].
Cell Isolation
Midluteal phase (9–10 days postovulation) endometrial tissue was obtained from adult female baboons by endometrectomy or after hysterectomy. Human endometrial tissue was obtained from endometrial biopsies or at hysterectomy from premenopausal women with no clinically documented abnormalities of the endometrium. All animal studies and the experimental use of human tissues were approved by the Animal Care Committee and the Institutional Review Board at the University of Illinois at Chicago, respectively.
The tissue was minced thoroughly in calcium- and magnesium-free Hanks' Balanced Salt Solution (HBSS). The minced tissue was placed in an enzyme solution containing 0.5% collagenase and 0.02% DNase and incubated for 20 min at 37°C in a shaking water bath. The supernatant was recovered and placed at 4°C. The remaining tissue was further digested in an enzyme solution consisting of 0.5% collagenase, 0.02% DNase, 0.1% hyaluronidase, and 0.1% pronase and processed as described above. The cell suspensions from the first and second digestions were centrifuged at 1500 x g for 5 min, and the pellet was resuspended in HBSS. The suspensions were then passed through a 95-µm nylon mesh followed by a 20-µm nylon mesh (Small Parts Inc., Miami, FL). The filtrates were pooled and centrifuged again at 1500 x g for 5 min. The pellet was resuspended in 30% Percoll solution and overlaid on top of a 60% Percoll solution. After centrifugation at 800 x g for 30 min, the cells that sedimented at the 30–60% interface were collected. The cells were washed twice in PBS supplemented with DNase and BSA. The cells were then resuspended in phenol red-free RPMI-1640 supplemented with 0.1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal calf serum depleted of steroids by treatment with dextran-coated charcoal. Cells were seeded at 1 x 105 cells/cm2 in 75-cm2 tissue culture flasks and incubated overnight at 37°C and 5% CO2. The medium was changed the next day and subsequently every 3 days. At confluence, cells were trypsinized (trypsin/EDTA) and subcultured into 12-well tissue culture plates. The medium was changed every 3 days. Cell purity was assessed by immunocytochemistry using antibodies against cytokeratin (Dako, Carpenteria, CA), vimentin (Zymed, San Francisco, CA) and factor VIII (Dako). The purity of the stromal cell preparations used in these studies was > 97%.
Treatment of Cells
At approximately 80% confluency after first passage, the cell culture medium was changed to RPMI-1640 supplemented with sodium pyruvate, penicillin/streptomycin, 2% stripped fetal calf serum, with or without 36 nM estradiol-17ß, 1 µM MPA, and 100 ng/ml highly purified porcine relaxin (kindly provided by Dr. David Sherwood, University of Illinois, Urbana). The word "hormones" is used in this paper to include both the steroids (estradiol and MPA) and relaxin. For long-term dbcAMP treatment, 0.1 mM dbcAMP was added with the hormones continuously for 17 days, and the medium was changed every 2 days. Twenty-four hours prior to each treatment time point, the medium was changed to serum-free RPMI-1640 supplemented with sodium pyruvate and penicillin/streptomycin, with or without hormones, and with or without dbcAMP. For short-term dbcAMP treatment, cells were pretreated with hormones only and were exposed to 0.1 mM dbcAMP at 48 h prior to each sampling time point. At each time point, the medium was collected and the IGFBP-1 present in the culture medium was measured using an RIA kit (Diagnostic Systems Laboratories, Webster, TX). Prolactin present in the culture medium was also measured using an RIA kit. Although no prolactin was detectable in our samples, the antibody provided in the commercial kit was able to detect baboon prolactin in cytosolic extracts of baboon term decidua and also amniotic fluid, both of which have high levels of prolactin.
Cells were lysed with TriReagent (Molecular Research Center, Cincinnati, OH), and RNA and protein were extracted using the protocol provided by the manufacturer. Cellular protein content was measured using the Micro BCA protein assay kit (Pierce, Rockford, IL).
Immunoblotting for SMA
Proteins (7.5 µg) from cell extracts were separated by one-dimensional SDS-PAGE (10%). The proteins were then transferred to nitrocellulose membranes (MSI, Westboro, MA) and incubated with an SMA monoclonal antibody (Dako) at a 1:1000 dilution. Horseradish peroxidase-conjugated second antibody was used at a 1:3000 dilution, and the immunoreactive product was visualized using the enhanced chemiluminescence system (Pierce). Myometrial tissue extracts [9] were included as positive controls for SMA on all Western blots. Specificity of SMA induction was confirmed by using two other monoclonal antibodies against actin (kindly provided by Dr. James Lessard, Children's Hospital Research Foundation, Cincinnati, OH; [10].
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
The RNA isolated from cells was reverse transcribed, and the cDNA was subjected to PCR. As a control, an RNA preparation that was not reversed transcribed was also subjected to PCR to ensure that no genomic contamination was being amplified. For the detection of different cDNAs, primers were designed from nucleotide sequences provided by GenBank (Table 1).
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The H3.3 PCR product was used as the internal standard for all samples. In all tissues, H3.3 is a histone that is constitutively expressed [11] and that does not change over time or with hormone treatment.
RT was performed in a final volume of 20 µl with 1 µg RNA and 50 U/µl murine leukemia virus reverse transcriptase using the GeneAmp RNA PCR core kit (Perkin-Elmer, Irvine, CA). The mixture was incubated at 42°C for 30 min. The RT product was aliquoted equally into two tubes and primers were added (50 pmol/tube); the volume was adjusted to 40 µl, and PCR amplification was performed with 0.5 µl of 5 U/ml Taq polymerase (Perkin-Elmer) and 0.25 µl of 10 mCi/ml [32P]dCTP per tube. Amplification was allowed to occur for 24 cycles consisting of 94°C (1 min), 60°C (2 min), and 72°C (3 min) followed by 15 min of final extension at 72°C. Primers for H3.3, IGFBP-1, and SMA were added together in a single tube so that amplification of the cDNAs for H3.3, IGFBP-1, and SMA occurred in the same tube. Also, RI, RIß, RII, and RIIß cDNAs were amplified together in a single tube. A linear curve was plotted using number of cycles of amplification versus densitometric values of the PCR products, measured with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) (see Fig. 4C). The optimal number of cycles for amplification that fit within the linear range was chosen for each set of primers.
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The PCR products were electrophoresed in 1.5% agarose gels, and the gels were dried and exposed to film. Data were quantified by image analysis using the PhosphorImager.
Statistical Analysis
Data were analyzed by least-squares ANOVA using the SuperANOVA software package (Abacus Concepts, Inc., Berkeley, CA). Sources of variation included experiment (defined as tissues from different animals/women), treatment (presence or absence of hormones/dbcAMP), days of treatment, and their interactions. For short-term dbcAMP treatment experiments, orthogonal contrasts were used to compare the means of the various treatments (see Fig. 5).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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SMA
During the luteal phase of the menstrual cycle, SMA staining was absent in stromal fibroblasts of the baboon endometrium. Limited staining was evident in smooth muscle cells surrounding the arterioles and venules (Fig. 1A). Immunohistochemical localization of SMA was evident in the stromal cells of the simulated-pregnant baboon endometrium (Fig. 1C) whereas in the pregnant animal, SMA expression was much lower and almost undetectable as compared to that in the simulated-pregnant animal (Fig. 1E).
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Stromal cells separated by enzymatic digestion from both human and baboon endometrium did not express the SMA protein prior to plating (Fig. 2). However, when stromal cells were plated and grown in culture, SMA was produced, suggesting that the culture conditions are sufficient to induce the expression of this protein. This occurred in both baboon (Fig. 2A) and human (Fig. 2B) stromal cells.
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Decidualization of Baboon and Human Stromal Cells
In vivo, IGFBP-1 was absent in the stromal cells of cycling (Fig. 1B) and simulated-pregnant baboon endometrium (Fig. 1D). IGFBP-1 staining was apparent in endometrial stromal cells that had undergone decidualization at the trophoblast-maternal junction (Fig. 1F; arrow), suggesting that a conceptus factor is involved in inducing the expression of IGFBP-1 during decidualization in vivo.
In order to induce decidualization in vitro in stromal cells of the baboon and human endometrium, cells were treated with estradiol, MPA, relaxin, and dbcAMP. Prolactin and IGFBP-1 were measured in culture media as indicators of decidualization. Human stromal cells produced both IGFBP-1 and prolactin in response to hormones alone. Both proteins were detectable under our culture conditions by Day 6 of treatment and increased thereafter (Fig. 3, B and D). Baboon stromal cells, on the other hand, produced minimal levels of IGFBP-1 in response to hormones alone (Fig. 3A). These levels were too low to be detected by Western blot analysis (data not shown). These cells did not produce detectable quantities of prolactin in response to hormones either (Fig. 3C).
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It has been demonstrated previously that agents that stimulate the cAMP second messenger system increase the production of prolactin and IGFBP-1 in hormone-treated human stromal cells in culture [6, 12]. Human stromal cells produced higher levels of IGFBP-1 and prolactin protein in response to dbcAMP and hormones as compared to the levels obtained with hormones alone. Significant levels of IGFBP-1 were observed as early as 2 days after initiation of dbcAMP and hormone treatment (see Fig. 5B; p 0.01). As shown in Figure 3A, in baboon stromal cells, increases in IGFBP-1 levels were observed with dbcAMP alone at 12 and 17 days (1.16 ng/µg protein and 4.32 ng/µg protein, respectively) and with hormones alone at 12 and 17 days (4.31 ng/µg protein and 8.22 ng/µg protein, respectively) in comparison to basal levels with no treatment at 12 and 17 days (0.05 ng/µg protein and 0.08 ng/µg protein, respectively). Treatment with dbcAMP and hormones after 12 and 17 days caused an increase in IGFBP-1 production (31.21 ng/µg protein and 58.32 ng/µg protein, respectively; Fig. 3A) that was high enough to be detectable by Western blot analysis (data not shown). Minimal levels of IGFBP-1 protein were detected by RIA at 2 and 6 days in all treatment groups. Levels of IGFBP-1 produced by baboon stromal cells were much lower than those produced by human stromal cells. Finally, no prolactin protein was detected by RIA in the culture media of baboon stromal cells at any time (Fig. 3C). Similar results were observed in response to the other cAMP analogue, 8-bromoadenosine 3':5'-cyclic monophosphate, and prostaglandin E2, which increases levels of intracellular cAMP. These data rule out the potential involvement of butyrate, a metabolic by-product of dbcAMP, in stromal cell differentiation.
IGFBP-1 mRNA expression in cells was demonstrated by RT-PCR (Fig. 4). In human cells, bands corresponding to the IGFBP-1 PCR product were evident in hormone-treated cells and dbcAMP-treated cells, and, most prominently, in cells treated with hormones plus dbcAMP (Fig. 4B). Baboon stromal cells also expressed IGFBP-1 mRNA in response to either dbcAMP or hormones only (Fig. 4A). However, much higher levels of IGFBP-1 mRNA were produced in response to dbcAMP and hormones. Given that RT-PCR is a very sensitive technique, it was not surprising to detect mRNA in response to either dbcAMP or hormones alone, since IGFBP-1 protein was also detected by RIA with these treatments.
Our in vivo studies showed that cells expressing SMA did not express IGFBP-1, whereas cells that produced high levels of IGFBP-1 did not express SMA (Fig. 1; [9]). Figure 4A shows that baboon cells expressing high levels of IGFBP-1 mRNA after long-term hormone and dbcAMP treatment (+/+ Day 12 and 17) do not express SMA. Human stromal cells that produced high levels of IGFBP-1 also showed a decrease in SMA expression (Fig. 4B).
Short-Term dbcAMP Treatment
Human and baboon stromal cells were pretreated with hormones for 2, 12, or 17 days, and dbcAMP was added for the last 48 h of each time point. This was done in order to determine whether long- or short-term treatment with dbcAMP was required for decidualization and IGFBP-1 expression. In human stromal cells, levels of IGFBP-1 significantly increased in response to 48 h of dbcAMP treatment at both 2 and 12 days (Fig. 5B; p 0.01). In baboon cells, a significant increase in IGFBP-1 was observed in response to dbcAMP only after 17 days of hormone pretreatment (Fig. 5A; p 0.01). The quantity of IGFBP-1 produced by both human and baboon cells in response to short-term dbcAMP treatment (Fig. 5) was much lower than the levels obtained after long-term treatment with dbcAMP (Fig. 3). These data show that dbcAMP is able to act in a short-term manner to induce IGFBP-1 production when cells are pretreated with hormones. Furthermore, it is evident that cells at the initial stages of hormone treatment differ in their responsiveness to dbcAMP from cells subjected to long-term hormone pretreatment.
PKA Regulatory Subunits
One mechanism by which the action of cAMP can be regulated is by the controlled expression of the various PKA regulatory subunits: RI, RIß, RII, and RIIß. Human stromal cells expressed mRNAs for all four subunits (Fig. 6B). RI was the major subunit expressed, followed by RII and RIIß, with the weakest expression being that of RIß. There was no observable difference in the expression of these subunits between decidualized and nondecidualized cells. In baboon stromal cells, RIIß was the major subunit expressed (Fig. 6A). RI and RII were also expressed; however, no RIß was detected. Similar to the situation with human cells, no observable difference in expression was evident between decidualized and nondecidualized cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Evidence presented in this study suggests that the processes of decidualization in the human and baboon involve similar mechanisms. Both human and baboon stromal cells expressed SMA in culture and, in response to hormones and dbcAMP, high levels of IGFBP-1. Furthermore, stromal cells from the human and baboon were responsive to short-term dbcAMP treatment after pretreatment with hormones. Within each species, decidualized and nondecidualized cells did not differ in their expression of PKA regulatory subunit isoforms. Thus, these two species appear to possess similar metabolic pathways necessary for decidualization, but the process of decidualization differs in its sensitivity or degree of responsiveness to external stimuli. Human decidualizing stromal cells generally produced higher levels of prolactin and IGFBP-1 than baboon cells and were more responsive to dbcAMP.
In vivo, decidualizing stromal cells in the baboon express prolactin much later than IGFBP-1 [7, 13]. Prolactin was detectable by immunocytochemistry in decidualizing stromal cells at the implantation site on Day 32 of pregnancy and became prominent between Days 40 and 60 of pregnancy. This staining pattern correlated with mRNA expression [13]. In contrast, IGFBP-1 expression in decidualizing stromal fibroblasts was first evident at the implantation site between Days 21 and 25 of pregnancy [7]. Thus, the in vitro data may also reflect the delayed expression of prolactin as compared to IGFBP-1. It is possible that this differential expression of the two decidual proteins in the baboon is the result of the more programmed and delayed decidual response in the nonhuman primate in comparison to the human, as well as the potential requirement for a conceptus to complete the decidualization process [14, 15]. Although the baboon decidual cells do not synthesize IGFBP-1 and prolactin at comparable time points, the cells that express IGFBP-1 in early pregnancy can be morphologically characterized as being decidual cells [16].
Cytoskeletal proteins are important in mitosis, cell growth, and changes in cell shape and also for the regulation of protein secretion [17]. Alpha SMA is an important filamentous protein associated with the cytoskeleton. In the baboon, SMA is absent in the stromal fibroblasts of the endometrium throughout the menstrual cycle but becomes evident as early as Day 14 of pregnancy and Day 18 of simulated pregnancy [9]. Between Days 32 and 40 of pregnancy, the number of positively stained stromal fibroblasts decreases, particularly around the implantation site, and by Days 50–60 this staining disappears. We have shown in our in vitro system that cells begin to produce SMA shortly after they are put into culture. This may be due to factors present in the culture media or factors secreted by these cells to promote adhesion. It has been shown that transforming growth factor ß induces SMA expression in smooth muscle cells [18] and human breast gland fibroblasts [19]. It is also possible that the elongation of isolated stromal cells after plating involves a remodeling of the cytoskeleton, requiring expression of certain cytoskeletal proteins. Chick fibroblasts, upon spreading, become elongated and form stress fibers characterized by actin bundles and vinculin streaks [20]. These stress fibers are quite noticeable in human and baboon endometrial stromal cells grown in monolayer. Alternatively, cell-cell contact through cell surface adhesion proteins could trigger the polymerization of the actin cytoskeleton within the cell. One possible role for the appearance of SMA, other than initiating changes in cell shape, may be to decrease the proliferation process in these cells in preparation for differentiation. It has been shown that induction of SMA by transforming growth factor ß in smooth muscle inhibits proliferation, thus maintaining the cell in its contractile state [18]. Also, changes in actin or actin-binding proteins appear to be important for cellular differentiation of granulosa cells [21]. It would be interesting to determine whether the expression of SMA is necessary in order for stromal cells to decidualize or whether this process is an independent phenomenon unrelated to the decidualization process.
It has been shown in vivo that baboon stromal cells expressing SMA protein do not coexpress IGFBP-1 protein [9]. We have demonstrated in this study that when cells express high levels of IGFBP-1 after long-term dbcAMP and hormone treatment, the levels of SMA mRNA expression decrease. It is possible that the expression of SMA or other cytoskeletal proteins is inhibitory to the expression of IGFBP-1 and that prolonged treatment with dbcAMP and hormones causes the disappearance or breakdown of cytoskeletal elements. Alternatively, it is possible that the cells must attain a critical level of IGFBP-1 in order to repress the expression of SMA. In cases in which SMA and IGFBP-1 mRNA were expressed simultaneously (Fig. 4A), it is probable that individual stromal cells in culture were at different stages of decidualization, with some cells producing only IGFBP-1 and others expressing only SMA. Further studies are required to clarify the relationship between IGFBP-1 and SMA expression.
The function of IGFBP-1 produced by the decidua is poorly understood. The IGFBPs either can enhance or inhibit insulin-like growth factor (IGF) action [22] or could act independently of IGFs and bind to integrins through the IGFBP-1 RGD sequence [23]. The involvement of IGFBP-1 in controlling trophoblast invasion has been suggested but remains unresolved. One possible mechanism supporting this concept involves the interaction of IGFBP-1 with integrins. It has been shown that mouse primary trophoblast cells when cultured on extracellular matrix proteins appear to interact with fibronectin exclusively through the RGD recognition site [24]. IGFBP-1 contains the RGD tripeptide sequence [25, 26], which can bind to the 5ß1 integrin receptor [23]. Thus, it is possible that maternal IGFBP-1 may affect the interaction between placental trophoblast and extracellular matrix proteins in the endometrial stroma and modulate invasion into the maternal decidua. If IGFBP-1 is indeed involved in controlling the invasive process of the trophoblast, it would be logical to assume that high levels of this protein in the decidua would be required to regulate the highly invasive nature of the human trophoblast. The extravillous trophoblasts have been occasionally found as deep as the myometrial layer of the uterus [27]. Invasion of the baboon trophoblast, as with most primates, is more superficial than that of the human and exhibits a limited area of decidualization that corresponds to areas of trophoblast contact [15]. Thus, the levels of IGFBP-1 required to regulate the invasion process in this species would be expected to be lower than the level in the human.
Several reports have indicated that stimulation of the cAMP signaling pathway is an important factor in decidualizing stromal cells [5, 6, 12]. The mechanism by which this is accomplished, however, requires further investigation. Our studies suggest that decidualization, as assessed by IGFBP-1 and prolactin induction, requires several intermediate steps associated with the cAMP signaling pathway. The response to cAMP was not immediate; it required 48 h of stimulation. This implies that after cAMP activation, additional changes occur within the stromal fibroblasts that in turn initiate the transcription of IGFBP-1 and prolactin. Our preliminary studies suggest that this process requires de novo protein synthesis (unpublished results). It has been well documented that stimulation of the cAMP pathway can affect transcription of certain genes. This occurs through proteins, such as cAMP response element (CRE)-binding protein, CRE modulators (CREM), and inducible cAMP early repressor (ICER), that specifically bind to the CRE region of the promoters. It has recently been shown that during the course of decidualization, human endometrial stromal cells express novel isoforms of CREM as well as exhibiting an up-regulation of ICER [28]. Whether these proteins are essential to the decidualization process remains to be elucidated.
Expression of mRNA of PKA subunits did not differ in nondecidualized and decidualized cells in either the baboon or the human. This confirms a recent study showing that all four isoforms were present in human endometrial stromal cells, with no difference in the mRNA expression of the isoforms between decidualized and nondecidualized cells [29]. Even the mRNA for catalytic subunits did not change. In contrast, Knutsen et al. [30] demonstrated that a mouse Sertoli cell line expressed the RI, RII, and RIIß subunits and that RIIß was up-regulated by cAMP, reaching maximal levels after 12 h of stimulation. In primary cultures of rat Sertoli cells, mRNAs for RI, RII, and RIIß are regulated with cAMP treatment [31] whereas in the TM4 cell line, none of the various PKA subunit mRNAs were regulated by cAMP. In ovarian follicular cells, RI protein levels increase while mRNA levels do not change in response to elevated cAMP levels [32]. Thus, it appears that PKA regulatory subunits are differentially regulated depending on the cell type.
In summary, it is apparent that the decidualization processes in humans and baboons involve similar molecules and pathways. It is also evident from our studies that cAMP in conjunction with hormones augments this differentiation process. Given the limitations on studies with early-gestational tissues from women, the baboon provides a valuable model for studying cellular regulation during early pregnancy. As such, functional studies done in vitro can be extrapolated to an in vivo system. The establishment of an in vitro decidualization model in a nonhuman primate will enable us to elucidate the precise biochemical and molecular events associated with this critical process required for the establishment of pregnancy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: A.T. Fazleabas, Dept. Obstetrics and Gynecology, University of Illinois at Chicago, 820 S Wood St, M/C 808, Chicago, IL 60612. FAX: (312) 996-4238; asgi{at}uic.edu
Accepted: March 2, 1998.
Received: September 3, 1997.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Z. Strakova, P. Mavrogianis, X. Meng, J. M. Hastings, K. S. Jackson, P. Cameo, A. Brudney, O. Knight, and A. T. Fazleabas In Vivo Infusion of Interleukin-1{beta} and Chorionic Gonadotropin Induces Endometrial Changes that Mimic Early Pregnancy Events in the Baboon Endocrinology, September 1, 2005; 146(9): 4097 - 4104. [Abstract] [Full Text] [PDF]
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A. Jasinska, V. Han, A. T. Fazleabas, and J. J. Kim Induction of Insulin-Like Growth Factor Binding Protein-1 Expression in Baboon Endometrial Stromal Cells by Cells of Trophoblast Origin Reproductive Sciences, September 1, 2004; 11(6): 399 - 405. [Abstract] [PDF]
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Z. Strakova, M. Szmidt, S. Srisuparp, and A. T. Fazleabas Inhibition of Matrix Metalloproteinases Prevents the Synthesis of Insulin-Like Growth Factor Binding Protein-1 during Decidualization in the Baboon Endocrinology, December 1, 2003; 144(12): 5339 - 5346. [Abstract] [Full Text] [PDF]
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 G. Zimmermann, D. Baier, J. Majer, and H. Alexander Expression of beta hCG and alpha CG mRNA and hCG hormone in human decidual tissue in patients during tubal pregnancy Mol. Hum. Reprod., February 1, 2003; 9(2): 81 - 89. [Abstract] [Full Text] [PDF]
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J. J. Kim, H. S. Taylor, G. E. Akbas, I. Foucher, A. Trembleau, R. C. Jaffe, A. T. Fazleabas, and T. G. Unterman Regulation of Insulin-Like Growth Factor Binding Protein-1 Promoter Activity by FKHR and HOXA10 in Primate Endometrial Cells Biol Reprod, January 1, 2003; 68(1): 24 - 30. [Abstract] [Full Text] [PDF]
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 N. Sugino, A. Karube-Harada, A. Sakata, S. Takiguchi, and H. Kato Different mechanisms for the induction of copper-zinc superoxide dismutase and manganese superoxide dismutase by progesterone in human endometrial stromal cells Hum. Reprod., July 1, 2002; 17(7): 1709 - 1714. [Abstract] [Full Text] [PDF]
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N. Sugino, A. Karube-Harada, S. Kashida, S. Takiguchi, and H. Kato Differential regulation of copper-zinc superoxide dismutase and manganese superoxide dismutase by progesterone withdrawal in human endometrial stromal cells Mol. Hum. Reprod., January 1, 2002; 8(1): 68 - 74. [Abstract] [Full Text] [PDF]
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A. Karube-Harada, N. Sugino, S. Kashida, S. Takiguchi, H. Takayama, Y. Yamagata, Y. Nakamura, and H. Kato Induction of manganese superoxide dismutase by tumour necrosis factor-{alpha} in human endometrial stromal cells Mol. Hum. Reprod., November 1, 2001; 7(11): 1065 - 1072. [Abstract] [Full Text] [PDF]
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N. Sugino, A. Karube-Harada, S. Kashida, S. Takiguchi, and H. Kato Reactive oxygen species stimulate prostaglandin F2{alpha} production in human endometrial stromal cells in vitro Hum. Reprod., September 1, 2001; 16(9): 1797 - 1801. [Abstract] [Full Text] [PDF]
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Z. Strakova, S. Srisuparp, and A. T. Fazleabas Interleukin-1{beta} Induces the Expression of Insulin-Like Growth Factor Binding Protein-1 during Decidualization in the Primate Endocrinology, December 1, 2000; 141(12): 4664 - 4670. [Abstract] [Full Text] [PDF]
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S. Banaszak, A. Brudney, K. Donnelly, D. Chai, K. Chwalisz, and A. T. Fazleabas Modulation of the Action of Chorionic Gonadotropin in the Baboon (Papio anubis) Uterus by a Progesterone Receptor Antagonist (ZK 137.316) Biol Reprod, September 1, 2000; 63(3): 820 - 825. [Abstract] [Full Text]
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N. Sugino, S. Kashida, S. Takiguchi, Y. Nakamura, and H. Kato Induction of superoxide dismutase by decidualization in human endometrial stromal cells Mol. Hum. Reprod., February 1, 2000; 6(2): 178 - 184. [Abstract] [Full Text] [PDF]
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A. Einspanier, R. Nubbemeyer, S. Schlote, M. Schumacher, R. Ivell, K. Fuhrmann, and A. Marten Relaxin in the Marmoset Monkey: Secretion Pattern in the Ovarian Cycle and Early Pregnancy Biol Reprod, August 1, 1999; 61(2): 512 - 520. [Abstract] [Full Text]
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J. J. Kim, J. Wang, C. Bambra, S. K. Das, S. K. Dey, and A. T. Fazleabas Expression of Cyclooxygenase-1 and -2 in the Baboon Endometrium during the Menstrual Cycle and Pregnancy Endocrinology, June 1, 1999; 140(6): 2672 - 2678. [Abstract] [Full Text]
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 A. T. Fazleabas, K. M. Donnelly, S. Srinivasan, J. D. Fortman, and J. B. Miller Modulation of the baboon (Papio anubis) uterine endometrium by chorionic gonadotrophin during the period of uterine receptivity PNAS, March 2, 1999; 96(5): 2543 - 2548. [Abstract] [Full Text] [PDF]
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J. J. Kim, R. C. Jaffe, and A. T. Fazleabas Insulin-Like Growth Factor Binding Protein-1 Expression in Baboon Endometrial Stromal Cells: Regulation by Filamentous Actin and Requirement for de Novo Protein Synthesis Endocrinology, February 1, 1999; 140(2): 997 - 1004. [Abstract] [Full Text]
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