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Regular Article |
a Unità Operativa di Ginecologia ed Ostetricia,
b Ospedale G.B. Grassi, Roma 00121, Italy Department of Obstetrics and Gynecology,
c Università Cattolica S. Cuore, Roma 00168, Italy Oasi Institute of Research,
d Troina, Enna 94018, Italy Department of Experimental Medicine and Pathology, Università La Sapienza, Roma 00185, Italy
ABSTRACT
The coculture of endometrial epithelial cells (EEC) with stromal cells (ESC) allows achievement of an improved in vitro system for studying interactions between cells via soluble signals. The purpose of this study was to investigate whether 17ß-estradiol and insulin can induce proliferation of EEC through ESC-secreted factors. No evidence of estrogen-induced EEC proliferation has been reported so far in the conventional culture methods. To this end, we used an in vitro bicameral coculture model where human EEC were grown on extracellular matrix-coated inserts applied in dishes containing ESC. Proliferation was assessed by tritiated thymidine incorporation. Homogeneity of endometrial cell populations was ascertained immunocytochemically. 17ß-Estradiol did not induce any proliferative effect on EEC cultured alone. Endometrial epithelial cell proliferation was significantly enhanced in EEC/ESC cocultures; moreover, it was further increased by 17ß-estradiol addition. Insulin increased proliferation in EEC cultured alone, but again the effect was more pronounced in EEC/ESC cocultures. Coincubation of 17ß-estradiol and an antibody against insulin-like growth factor I (IGF I) led to neutralization of ESC-mediated EEC proliferation. This work provides evidence that the effect of 17ß-estradiol on human EEC proliferation may be mediated at least in part through ESC-secreted IGF I. We also showed that insulin effect is also partially due to ESC activation.
growth factors, hormone action, uterus
INTRODUCTION
The attention of a growing number of investigators has been focused on the potential role of secreted factors in modulating cell-cell communications involved in epithelial growth and maturation. These factors have been only partially characterized, but it has been suggested that some could represent a biochemical means of interplay allowing cells to communicate with one another, in a paracrine and/or autocrine fashion, to direct proliferative and maturational events [1]. This theory is supported by several observations made in different tissues: it is known that certain androgenic effects upon prostatic epithelial cells are mediated via stromal cells and that the stroma of mammary gland is able to support both differentiation of epithelium in vivo and growth of epithelial cells in vitro [2–4].
Notwithstanding that this field of investigation is even more complex in the endometrium, due to the continuous modifications and renewal this tissue undergoes cyclically, a considerable amount of experimental evidence supports the relevance of epithelial-stromal relationships even at this level [5–7]. This need for cell-cell interactions could explain the lack of responsiveness to steroid hormones and growth factors observed by many authors in cultured endometrial epithelial cells (EEC) [8–10]. In fact, while proliferation in response to exogenous estrogen is well proved in vivo, it has been exceedingly difficult to demonstrate even minor mitogenic reponses to estrogens in isolated human or rodent EEC in culture. It seems that essential mediators for EEC responsiveness to estrogens are missing in the monolayer culture system. Indeed, proliferation of EEC might be induced by putative growth factors or inductors elaborated in the neighboring stromal cells (ESC) in response to hormonal stimulation. Insulin is thought to be an important regulator of EEC proliferation, as several reports have shown its direct proliferative effect on EEC [8, 9], and women with hyperinsulinemia are more likely to develop endometrial hyperplasia [11]. It is also known that insulin can locally regulate locally the activity of the insulin-like growth factor system by decreasing the binding protein-1 (IGF BP-1) levels, thus increasing insulin-like growth factor I (IGF I) availability [12].
In order to study epithelial-stromal relationships in human endometrium, and to investigate whether 17ß-estradiol and insulin could induce proliferation of EEC through an indirect mechanism involving ESC, we developed an in vitro coculture model that appears to preserve at least some of the paracrine interactions supporting EEC responsiveness. Our model, in which EEC are grown on extracellular matrix (ECM)-coated filters, may provide a more suitable tool for studying endometrial physiology than separate culture of EEC and ESC. We also attempted to clarify the mechanisms underlying the influence of ESC on EEC proliferation by use of antibodies against specific growth factors.
MATERIALS AND METHODS
Chemicals and Culture Supplies
Dulbecco modified Eagle medium (DMEM), both regular and phenol red-free, Hanks balanced salt solution without calcium and magnesium, trypsin, and fetal calf serum (FCS) were purchased from Gibco BRL (Parsley, Scotland). Collagenase IA, insulin (from bovine pancreas), transferrin, 17ß-estradiol, and the monoclonal antibodies against IGF I and epidermal growth factor (EGF) were obtained from Sigma (St. Louis, MO). Basement membrane matrix (Matrigel) was purchased from Collaborative Research (Bedford, MA). Plastic materials, including centrifuge tubes, T25 cm2 culture flasks, 24-well polyethylene cell culture inserts (0.4 µm pore size), and companion 24-well plates were purchased from Falcon (Oxnard, CA). [3H]Thymidine was obtained from Amersham (Milano, Italy).
Tissues
Endometrial specimens were obtained from normally cycling women, aged between 23 and 40 yr, undergoing operative laparoscopy for nonendometrial problems (subserous uterine fibromata, pelvic pain). Patient's informed consent to use uterine tissue for experimental purposes was obtained before surgery in all cases. Biopsies were obtained by courettage of the uterine cavity. A portion of each sample was fixed in 4% formalin and processed for histologic dating, following Noye's criteria [13]. All tissue samples considered for this study were from the late proliferative phase (11th to 14th days). Evidence of dominant follicle was also reported during laparoscopic examination.
Isolation and Primary Culture of EEC and ESC
To perform the whole study, a total of 18 endometrial specimens from the late proliferative phase were processed for separation of EEC and ESC. The EEC and ESC were separated following the method described by Satyaswaroop [14] with minor modifications. Briefly, minced endometrial tissue was digested with 0.25% collagenase in 10% FCS DMEM for about 1 h. Digestion was repeatedly checked under an inverted microscope until isolated glands and single cells could be visualized. The suspension was then filtered through a 250-µm pore size sieve (Tecnochimica Moderna; Monterotondo, Roma, Italy), and transferred to a conical tube that was left in upright position for a few minutes, in order to allow glands to settle under gravity. Single cells in the top two thirds (among which are the ESC) were filtered through a 40-µm cell strainer (Falcon, Oxnard, CA) and centrifuged twice for 10 min at 1200 rpm, while the bottom 2 ml, containing the glandular pellet, was resuspended in fresh DMEM. The last step was repeated several times. The EEC and ESC were then seeded separately in plastic tissue culture flasks (T25 cm2) in phenol red-free DME nutrient medium (containing 2% of antibiotic-antimycotic mixture [6.3 g/L penicillin, 10 g/L streptomycin sulfate, 250 ng/mL fungizone], 10 µg/mL insulin, 25 µg/mL transferrin, and 10% FCS) and incubated at 37°C in a humidified atmosphere of 5% CO2 in air. The ESC were washed and medium was replaced as soon as cells attached (<30 min) in order to get rid of the few epithelial contaminants. Medium was changed 24 h after seeding and renewed at intervals of 48 h until EEC and ESC primary cultures achieved subconfluence.
Preparation of Matrigel-Coated Inserts and Subculture of EEC and ESC
Matrigel-coated inserts were prepared as described by others [15]. Briefly, precooled culture inserts were placed into 24-well culture dishes and coated with 50 µl of 1:8 diluted Matrigel in cold phenol red-free DMEM (phenol red has a weak estrogenic activity). Inserts were kept for about 2 h at room temperature, excess liquid was aspirated, and culture surfaces were air dried under a laminar flow hood and washed twice before use. Subconfluent cultures of EEC and ESC were submitted to trypsinization, sedimented by centrifugation, and resuspended in fresh medium. Cellular suspensions containing EEC (75 000 cells) were then applied to Matrigel-coated inserts. The ESC were subcultured into a separate 24-well plate at a density of 100 000 ESC/well. Sufficient quantities of both EEC and ESC were harvested for each separation procedure to ensure a set of three replicates of coculture bicameral dishes for each treatment. The EEC and ESC were cultured in plain phenol red-free DMEM (without insulin, transferrin, and FCS) before challenge with hormone treatment after 48 h. Some EEC and ESC were collected on glass slides for parallel immunocytochemical procedures and assessment of cell purity. After 48 h, Matrigel-coated filters with EEC were inserted into culture dishes containing confluent ESC. In the mean time, medium was replaced both in the basal and apical compartments with fresh medium containing treatments and 3H-thymidine (4 µCi/ml). In a second series of experiments, a monoclonal antibody against IGF I (2.5 µg/ml) or against EGF (0.1 µg/ml) was added to media together with estradiol.
Dose-dependency studies for 17ß-estradiol (10-6–10-8 M) and insulin (1–100 µg/ml) were also performed both in EEC cultured alone and cocultured EEC/ESC.
After 24 h of culture, cell proliferation was assessed by 3H-thymidine incorporation.
The EEC cultured on Matrigel-coated filters (without the application of ESC in the basal compartment) were considered as controls to assess ESC-induced growth. In the presence of treatments, increases were compared to values obtained in EEC/ESC cocultures.
Cell Proliferation
Cell proliferation, as reflected by DNA synthesis, was measured in the apical chamber by the incorporation of 3H-thymidine, following a previously described procedure with some modifications [16]. Briefly, once the radioactive medium was removed, EEC were washed twice with ice-cold PBS, twice with 5% trichloroacetic acid (TCA), and once with 10% TCA. Cells were then lysed with 1 N NaOH, and the reaction was finally stopped with 37% HCl. Two hundred microliters from each insert were counted for 5 min in 5 ml scintillation liquid (Packard Instruments Co., Milano, Italy). A blank (a Matrigel-coated insert without cells) was included in all 3H-thymidine incorporation determinations and the resultant values subtracted from the ones from cell-containing inserts. In a separate set of culture wells in three different experiments, cells were trypsinized and counted after coculture in a hemacytometer in order to assess cell number.
Insulin-Like Growth Factor I Determination
In order to determinate IGF I release by ESC, after 4 days primary culture cells (100 000 ESC/well) were trypsinized and subcultured in 24-well culture dishes for 24 h both in basal conditions and in the presence of 17ß-estradiol (10-7 M) or insulin (10 µg/ml). The IGF I content in culture media was assayed by the use of a commercially available RIA kit (IGF I RIA kit; Nichols Institute Diagnostic, S. Juan Capistrano, CA). The sensitivity of the assay was 0.06 ng/ml, and its inter- and intraassay coefficients of variation were <8.4% and <3%, respectively. Cross-reactivity with insulin was <0.01%.
Evaluation of Cell Purification
Cell purity was evaluated immunocytochemically as previously described [17]. The EEC and ESC spun on glass slides were immediately fixed in 95% ethanol and immunostained for cytokeratin, vimentin, and FIII-related antigen, using an avidin-biotin complex (ABC) immunoperoxidase kit (Vectastatin Elite ABC kit; Vector Labs, Burlingame, CA). Diaminobenzidine was used as the chromogen and hematoxylin as nuclear counterstain. As primary antibodies, mouse monoclonal anti-human cytokeratin and vimentin and rabbit polyclonal anti-FIII-related antigen were used (Ylem; Avezzano, Aquila, Italy). Negative controls (nonimmune serum) were included in all immunoreactions. Immunoreactivity was evaluated in all experiments, by examining multiple fields for the percentage of reactive cells. The mean percentage of reactive cells was calculated by dividing the number of positive cells by the total number of cells counted for each slide.
Statistical Analysis
Data were analyzed by repeated-measures ANOVA and subsequent Tuckey-Kramer's test for multiple comparisons. The linear coefficient between cell number and 3H-thymidine incorporation (R) has been calculated by Pearson test for parametric correlations.
RESULTS
Cell Morphology and Cell Purification
Isolation and purification of cells from uterine courettings yielded a variable number of cells that ranged between 10–30 x 106 EEC and 10–20 x 106 ESC. Purification of cells resulted in recovery of about 75% of the cell suspension generated after the initial dissociation step. Cell viability was >90% and 80% for EEC and ESC, respectively. After cell dissociation, isolated glands were observed as tubular structures. Twenty four hours after seeding, a monolayer of cells started growing out radially from glandular explant (Fig. 1A). After 72–96 h of culture, EEC appeared as a subconfluent monolayer that consisted of cells polyhedral in shape and typically epithelioid (Fig. 1B). The ESC were mainly isolated round cells that grow as spindle-shaped cells with long cytoplasmic processes. They usually reached subconfluence after 72 h of culture, displaying a single cell monolayer growth pattern (Fig. 1C).
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The EEC subcultured on Matrigel formed scattered isles of cells that do not reach confluence and did not appear to flatten on culture surface, as also shown by Shatz et al. [18] (Fig. 1D).
Immunocytochemical staining with the use of specific monoclonal antibodies was used to evaluate the cell purification method. Cells were examined after primary culture (4 days), before the establishment of coculture. Antiserum to cytokeratin reacted intensely with EEC and revealed the characteristic cytoarchitecture of the intermediate filaments cytokeratins (Fig. 2A). The percentage of immunoreactive EEC was >95% of total cells in all cases. Only few cells were found to express vimentin (<8%). Conversely, the anticytokeratin antiserum did not react with ESC (Fig. 2B); in fact, >90% of ESC were found negative for cytokeratin while most of them stained with antivimentin antibody (>90%). Monoclonal antibody to FVIII-related antigen reacted only with a few cells (<2–3%) in some ESC cultures. Cells positive for FVIII were only rarely seen in EEC cultures.
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Immunocytochemistry of EEC performed after coculture did not show any difference in cell purity compared to staining obtained before coculture, thus ruling out the possibility of selective growth of the few contaminant cells.
3H-Thymidine Incorporation
The results of the 3H-thymidine incorporation assay are shown in Figures 3 and 4. Incorporation of 3H-thymidine as a reflection of deoxyribonucleic acid synthesis is reported as counts per minute (cpm; mean ± SEM of triplicate measurements of five different experiments, n = 5).
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The EEC basal 3H-thymidine incorporation, obtained in EEC cultured alone (158.8 ± 35.6 cpm) was significantly enhanced when ESC were cocultured in the basal compartment (219.9 ± 43.4 cpm, 38% increase). Moreover, although no difference was noted between EEC cultured alone in basal conditions and in the presence of 17ß-estradiol 10-7 M/L (158.8 ± 35.6 vs. 162.4 ± 34.2 cpm), in EEC/ESC cocultures 17ß-estradiol was able to strongly induce 3H-thymidine incorporation in the former cells (315.8 ± 57.3 cpm) with respect to both EEC basal incorporation (98% increase) and the level observed in EEC/ESC coculture (43% increase). These data suggest that ESC are able to induce EEC 3H-thymidine incorporation, and that this effect can be further increased by the addition of 17ß-estradiol.
In a different set of experiments the effect of insulin on ESC-mediated EEC proliferation was investigated. Results are displayed in Figure 4. Insulin (10 µg/ml) by itself was able to induce a significant enhancement of 3H-thymidine incorporation in EEC cultured alone (104.9 ± 11 cpm vs. 74.6 ± 5 cpm, corresponding to a 36% increase); however, the addition of insulin to EEC/ESC coculture was followed by a stronger increase in EEC 3H-thymidine incorporation (148.7 ± 6 cpm) with respect to both EEC cultured alone (104.9 ± 11 cpm, 41% increase) and EEC cocultured with ESC (116.6 ± 7 cpm, 7% increase) subjected to the same treatment.
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In order to determine whether 17ß-estradiol and insulin proliferative effects on EEC were dose-related, EEC were cultured in parallel alone on Matrigel-coated inserts or cocultured with homologous ESC in the basal compartment in the presence of increasing concentrations of 17ß-estradiol (10-6–10-8 M/L) and insulin (1–100 µg/mL).
The addition of 17ß-estradiol in the range of 10-6–10-8 M/L did not modify the level of 3H-thymidine incorporation in EEC when cultured alone (Table 1), while it induced a dose-dependent increase when EEC were cocultured with ESC.
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On the other hand, EEC cultured with increasing doses of insulin (1–100 µg/ml) showed a similar trend toward a dose-dependent stimulation of 3H-thymidine incorporation rate, either when cultured alone or cocultured with ESC (Table 1).
Cell count after coculture showed an increase in cell number in keeping with 3H-thymidine incorporation (R = 0.998) (Table 2).
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Neutralization Studies
In order to characterize the possible paracrine factor(s) responsible for the ESC-mediated EEC proliferation, we set up a series of experiments in which specific antibodies against two of the most important growth factors that are likely involved in endometrial growth (i.e., IGF I and EGF) were added to cocultured endometrial cells. The effect of the above mentioned antibodies was evaluated both in basal and 17ß-estradiol-stimulated conditions. The results of the neutralization studies are shown in Figure 5, as means ± SEM of triplicate measurements of three different experiments (n = 3). Both anti-IGF I and anti-EGF antibodies appeared to induce a slight, but not statistically significant, decrease of basal (not stimulated) ESC-induced EEC proliferation (Fig. 5A). Proliferation of EEC in this series of experiments was again significantly enhanced by coculture with ESC (182.7 ± 45.2 vs. 131.1 ± 27.4, 37% increase).
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In Figure 5B, the effects of the antibodies against IGF I and EGF on EEC/ESC coculture in the presence of 17ß-estradiol are shown. The concomitant treatment of cocultured endometrial cells with the antibody anti-IGF I and 17ß-estradiol led to a complete neutralization of the significant ESC-mediated EEC proliferation induced by 17ß-estradiol (136.3 ± 42.9 vs. 248.6 ± 74.5, 54%). Only a slight, not significant reduction was observed after concomitant treatment of EEC/ESC coculture with 17ß-estradiol and the anti-EGF antibody. As also observed in EEC/ESC cocultures, both used monoclonal antibodies slightly modified basal proliferation of EEC cultured alone (139 ± 28 and 128 ± 30 vs. 158.8 ± 70 cpm for anti-IGF-I and anti-EGF antibody, respectively), suggesting that these antibodies may be blocking a slight autocrine regulation of EEC or a little stimulation due to growth factors eventually present in the culture medium.
Insulin-Like Growth Factor I Release by ESC
Treatment of ESC with 17ß-estradiol (10-7 M/L) caused an increase in IGF I release compared to ESC in basal medium (1.83 ± 0.23 vs. 3.1 ± 0.98 ng/ml, respectively, P < 0.05). Insulin also induced a marked increase in IGF I release by ESC compared to control (5.1 ± 2.1 ng/ml, P < 0.05).
DISCUSSION
Estrogen responsiveness is a complex phenomenon involving several factors. Most cell types in the uterus demonstrate a certain degree of estrogen responsiveness in vivo. The most dramatic changes occur in the luminal and glandular epithelium during the follicular phase of the cycle, when these issues depend on estrogen for proliferation. In contrast, uterine stromal and myometrial layers appear to be affected minimally, in terms of morphology, by estrogens. In contrast to this responsiveness in vivo, human and rodent endometrial epithelial cells are either unresponsive or demonstrate only a poor response to estrogen in vitro, requiring high concentrations to achieve minimal responses [6, 8–10, 19]. Many hypotheses have been envisaged to explain such a discrepancy. Initially, some authors speculated that EEC responsiveness to estrogens could be mediated by estromedins, that is to say substances secreted elsewhere under the effect of estrogens and carried to the uterus by blood circulation [20]. This theory is supported by in vitro evidence for the requirement of serum for estrogen to induce proliferation in pig EEC [8]. However, this is not likely, because in vivo the upper layer of endometrium does not necessarily come into contact with many serum substances [20]. An estrogen receptor deficiency (either qualitative or quantitative), due to culture of EEC on plastic surfaces, had also been suggested [21]. In the effort to establish appropriate culture conditions, EEC have also been cultured under polarizing conditions on ECM, in order to maintain a cytoarchitecture resembling that of EEC in vivo [18]. In fact, the contact with components of ECM is believed to be important for the expression of differentiated functions in several systems [22]. Even under these culture conditions and despite the functional integrity of receptor system [9], 17ß-estradiol was unable to induce EEC proliferation [19]. Recent studies suggest that paracrine growth factors and stromal-epithelial interactions are necessary components of EEC proliferative response in different tissues [23]. Cooke et al. [5] showed that EEC regained estrogen responsiveness only when transplanted in vivo in combination with appropriate stroma. Moreover, in estrogen receptor knockout mice, they also recently demonstrated that EEC estrogen receptor is not necessary for estradiol-induced growth, and that the estrogen receptors on ESC mediate mitogenic effects on uterine epithelium [24]. The present study demonstrates the efficacy of epithelial-stromal cocultures as a tool for studying paracrine interactions between endometrial cells because the results of this study clearly show that 17ß-estradiol is able to induce proliferation of EEC by acting through ESC and/or in concert with an ESC-derived factor. Data from 17ß-estradiol and insulin challenges display a different response of EEC to stimulation, suggesting potentially different mechanisms of action. In fact, insulin and ESC have an additive effect on EEC 3H-thymidine incorporation, while 17ß-estradiol and ESC induce an evident synergistic response, suggesting that ESC are able to respond to 17ß-estradiol with increased DNA synthesis in EEC.
Our data are not in keeping with other reports from animal models [6, 25], in which the authors suggested that EEC-ESC contact is critical for 17ß-estradiol stimulation [6]. In another in vitro model of neonatal mouse uterus, Everett et al. concluded that uterine mesenchyme produces a diffusible factor that enhances the growth of epithelium, but that the addition of estrogen to epithelial-mesenchyme cocultures had no significant effect on cell number [26]. However ECM was not used in these earlier in vitro models. Further, differences do appear to exist between species regarding endometrial tissue differentiation [27]. Such species differences also may play a role in the differences between our present observations and previous reports by others. A number of growth factors and growth factor receptors have been identified in human and rodent endometrium. In particular, EGF and IGF I seem to be involved in endometrial physiology [28, 29], their effect being modulated by sex steroids. In fact, 17ß-estradiol modulates EGF signal through an alteration of EGF receptor expression [29]. Regarding the IGF I signal, the interactions seem to be more complex, because estrogens can modulate its activity both by enhancing ligand levels and receptor expression [30] and by decreasing IGF BP-1 [28].
The neutralization study data suggest that 17ß-estradiol may affect EEC proliferation by means of ESC-secreted IGF I rather than EGF. If this is the case, the modulation of IGF I signal could occur by an increase in IGF I synthesis and release and/or by a reduction of the secretion of IGF BP-1 by ESC [12].
These neutralization study data and the present observation that IGF I content increases in media from ESC cultures in response to estradiol provide strong support for the concept that IGF I may be involved in ESC paracrine regulation of estrogen action on EEC. Such a relevant role of IGF I in EEC growth has also been suggested by several studies [30–32].
Among the various polypeptide growth factors that are likely implicated in the EEC growth regulation, insulin is one of the most characterized because insulin receptors are present in human endometrium, showing a cyclic variation throughout the menstrual cycle [31]; moreover, their expression appears to be under the influence of steroid hormones [33].
In the present study, insulin directly affects EEC 3H-thymidine incorporation and potentially cellular proliferation. Moreover, insulin also affected EEC 3H-thymidine incorporation indirectly through the ESC. This ESC-related effect of insulin on EEC 3H-thymidine incorporation may involve, at least in part, the insulin-stimulated increase in IGF-I release by ESC that was also observed in this study. However, it is unlikely that, in the present conditions, the insulin-induced secretion of IGF I by ESC stimulated the EEC further, because the dose of insulin used is sufficient to saturate the IGF I receptor. Moreover, also because of the complex manner in which insulin regulates the expression of IGF BPs in endometrial tissue [12, 31, 33, 34], the extent to which insulin may be affecting EEC 3H-thymidine incorporation in the present study is not clear at this time.
In conclusion, our work provides direct evidence that the effect of 17ß-estradiol on human EEC proliferation may be mediated, at least partially, through ESC-secreted IGF I. Moreover, we also showed that the proliferative effect of insulin on EEC in vitro is partially mediated through ESC. In vitro systems are notorious for altering normal responses of many cells types to physiologic in vivo stimuli. However, the in vitro model developed for EEC, used in the present study, is the closest one to the in vivo situations, because it appears to have overcome the difficulties associated with EEC proliferation in vitro. The results of this work confirm the relevance of epithelial-stromal relationships in human endometrium and indicate that this in vitro system may be used to investigate further the role of paracrine interactions in the regulation of normal and abnormal tissue growth and function, such as endometrial hyperplasia, cancer, and anomalies of decidualization during the implantation process.
FOOTNOTES
First decision: 22 March 2000.
1 Support for this study was provided by a grant from Shering S.p.A., Milan, Italy. 
2 Correspondence: Antonio Lanzone, Istituto di Ginecologia ed Ostetricia, Università Cattolica S. Cuore, L.go A. Gemelli, 8, Roma 00168, Italy. FAX: 39 06 3051160; alanzone{at}rm.unicatt.it
Accepted: October 30, 2000.
Received: February 3, 2000.
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