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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 11 4006-4012
Copyright © 1998 by The Endocrine Society

Original Studies

Promegestone (R5020) and Mifepristone (RU486) Both Function as Progestational Agonists of Human Glycodelin Gene Expression in Isolated Human Epithelial Cells1

Robert N. Taylor, Jean-François Savouret, Christian Vaisse, Jean-Louis Vigne, Isabelle Ryan, Daniela Hornung, Markku Seppälä and Edwin Milgrom

Unité de Recherches Hormones et Reproduction (R.N.T., J-F.S., C.V., E.M.), INSERM U135, Université Paris-Sud, Hôpital de Bicêtre APHP, 94275 Le Kremlin-Bicêtre, France; Reproductive Endocrinology Center (R.N.T., J-L.V., I.P., D.H.), University of California, San Francisco School of Medicine, San Francisco, California 94143; and Department of Obstetrics and Gynaecology (M.S.), Helsinki University Central Hospital, Haartmaninkatu 2, SF-00290 Helsinki, Finland

Address all correspondence and requests for reprints to: Robert N. Taylor, M.D., Ph.D., Reproductive Endocrinology Center, University of California, San Francisco School of Medicine, San Francisco, California 94143-0132.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
One of the most abundant protein products of human secretory endometrium is glycodelin, a glycoprotein previously referred to as PP14. Although the precise function of this protein is unknown, its unique glycosylation pattern is believed to affect immunomodulatory activity during human embryonic implantation and inhibition of sperm-egg binding after ovulation. Having confirmed the expression of glycodelin in secretory endometrial glands, we used purified endometrial epithelial cell cultures to demonstrate the hormonal regulation of glycodelin synthesis and secretion. The findings were corroborated by transiently transfecting glycodelin gene promoter-reporter constructs into human epithelioid HeLa and Ishikawa cells. Our results indicate that glycodelin protein production by endometrial epithelial cells is directly up-regulated 4- to 9-fold by progestins and antiprogestins in vitro. Transcriptional regulation of the glycodelin gene promoter expressed in HeLa cells is progesterone receptor-dependent. As observed in the primary endometrial cells, progestins and antiprogestins both act as agonists on the in vitro expression of this endometrial gene. Our findings provide insight into the regulation of this abundant endometrial protein and raise interesting questions about the physical nature of the interaction of agonist- and antagonist-bound progesterone receptors with the glycodelin gene promoter.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE HUMAN endometrium undergoes a precisely orchestrated cyclical development and secretory differentiation in preparation for blastocyst implantation each month. Corpus luteum progesterone production is indispensable for the maintenance of early pregnancy in women (1), and progesterone receptor (PR) null mice confirm the requirement of its action for normal endometrial function in the rodent (2).

The identification and characterization of progesterone-induced uterine proteins, in the human and other eutherian mammals, have been a major focus in the quest to understand hormonal regulation of uterine receptivity and establishment of early pregnancy. Several specific proteins have been identified, including uteroglobin in the rabbit (3); ferritin heavy chain in the rat (4); and {alpha}vß3 integrin (5), mucin-1 (6), and glycodelin (7) in the human.

Glycodelin, originally identified by the misnomer: placental protein 14 (8), is a 28-kDa monomeric glycoprotein and the predominant product of human secretory endometrial epithelial cells. Though primarily produced in the uterus, expression of this protein also has been detected in seminal fluid (9) and in cultured hematopoietic precursor cells (10). Its complementary DNA was isolated first by Julkunen et al. (11); and shortly thereafter, the gene was cloned (12) and localized to human chromosome 9 (13). Analysis of the genomic structure and nucleotide sequence indicated that human glycodelin is related evolutionarily to the ß-lactoglobulin family of mammalian proteins. Functional studies of glycodelin are limited, but they indicate that glycodelin has immunomodulatory effects (14, 15) and can inhibit human sperm-oöcyte interaction in vitro (16). The latter effect seems to be mediated by oligosaccharide moieties. Glycodelin carries two sets of asparagine-linked high mannose, hybrid, and complex-type glycan structures (17). Its abundant in vivo expression in human secretory endometrium and decidua suggests that it plays an important role in embryonic implantation.

Glycodelin protein is expressed in secretory-phase endometrial epithelial glands of normally cycling women (18, 19). Circulating concentrations of glycodelin are low in the follicular phase of the cycle, but they rise when progesterone levels peak during the luteal phase (20). Plasma concentrations increase further in early pregnancy. Progestin-containing intrauterine devices prematurely induce the endometrial expression of glycodelin during the follicular phase of the ovulatory cycle (21). Postmenopausal women receiving estrogen and progestin have higher circulating glycodelin concentrations than those receiving estrogen alone (22). Thus, glycodelin expression can serve as a peripheral marker of the effects of progesterone on the uterine mucosa.

A prior attempt to demonstrate progestational regulation of glycodelin production by decidual explants in vitro failed to show a direct hormonal effect (23). Some investigators have invoked an obligatory role of nonsteroidal ovarian factors in the regulation of glycodelin (24, 25). In the current study, we confirmed the expression of glycodelin in human secretory endometrium, evaluated the effects of classical progestins and antiprogestins on the expression of glycodelin protein in isolated primary human endometrial cells in vitro, and investigated the transcriptional regulation of the human glycodelin gene promoter in transfected human epithelioid cells.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Tissue collection and evaluation

Endometrial tissue was collected by Pipelle aspiration biopsy from nonpregnant volunteers undergoing diagnostic laparoscopy. The patients were normally cycling women whose ovulatory status was confirmed by menstrual calendars, serial ultrasonography, and luteal-phase serum progesterone concentrations. All specimens were obtained from women who provided written informed consent under a protocol approved by the Committee of Human Research at the University of California, San Francisco.

Portions of the endometrial biopsies were fixed in 4% paraformaldehyde, embedded in paraffin, and cut as 5-µm sections for histochemical analyses. The menstrual cycle phases of these samples were estimated by the classical histological criteria (26). Midproliferative and midsecretory-phase endometrial biopsies were used for the isolation and preparation of endometrial epithelial and stromal cell cultures.

Hormones and chemicals

The progestin agonist promegestone (R5020, 17,21-dimethyl-19-norpregna-4,9-dien-3,20-dione) and the antagonist mifepristone (RU486, 17ß-hydroxy-11ß-(4-dimethylamino-phenyl)-17{alpha}-(1-propynyl)-estra-4,9-dien-3-one) were gifts from Dr. D. Philibert (Roussel-UCLAF, Romainville, France). Onapristone (ZK299, 11ß-(4-dimethylaminophenyl)-17{alpha}-hydroxy-17ß-(3-hydroxypropyl)-13{alpha}-methyl-4,9-gonadien-3-one) was a gift from Dr. H. Michna (Schering, Berlin, Germany). Progesterone, 17ß-estradiol, and other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO).

Glycodelin immunostaining

Immunohistochemistry was performed using the Vectastain kit according to the manufacturer’s specifications (Vector Laboratories, Inc., Burlingame, CA). The primary antibody was immunopurified rabbit antihuman glycodelin IgG, described below and used at a final concentration of 4 µg/mL. Nonimmune rabbit IgG was used at the same concentration as the control antibody.

Preparation of primary human endometrial cell cultures

Endometrial epithelial and stromal cells were cultured from endometrial biopsies, as described (27). Primary epithelial cells were cultured directly in 24-well plates. Stromal cells from the same biopsies were passaged twice, to eliminate contaminating immunocytes, and plated in 24-well plates. Cells were grown to confluence in MEM-{alpha} with nonessential amino acids, 10% FCS, and penicillin/streptomycin. Previous studies, using cytokeratin and vimentin immunostaining, respectively, verified that the epithelial and stromal cell cultures were more than 95% pure. Moreover, the cultures were free of T-cells, granulocytes, monocytes, and other leukocytes, as detected by CD3, CD11b, and CD45 immunostaining (27).

Metabolic labeling of endometrial cell products with [35S]methionine and [35S]cysteine

Culture media were changed to methionine- and cysteine-free, low-serum medium [MEM-{alpha} with nonessential amino acids, 2.5% FCS and penicillin/streptomycin, supplemented with 50–100 µCi/mL [35S]methionine and [35S]cysteine (Promix, Amersham, Arlington, IL)]. Conditioned media were collected after 18–24 h and were centrifuged at 15,000 x g for 5 min to sediment cells. The supernatants were frozen at -20 C. Progestin (R5020 and progesterone) and antiprogestin (RU486 and ZK299) treatments were performed at concentrations ranging from 0.01–100 nmol/L. The cells were metabolically labeled for up to 24 h. [35S]-Labeled endometrial cell proteins were separated by 10% SDS-PAGE under reducing conditions (5 mmol/L ß-mercaptoethanol), treated with diphenyloxazole in acetic acid, dried, and exposed to Kodak XAR film (Eastman Kodak, Rochester, NY) for 24–72 h. Glycodelin secretion was quantified by fluorography and computer-assisted densitometry of the 28-kDa bands (BioImage, Ann Arbor, MI). Relative absorbance units were normalized on a per-cell basis by the fluorimetric quantification of epithelial cell DNA in each well (28) and expressed as a ratio of untreated control cultures.

Immunoblot analyses

Endometrial cell culture supernatants were subjected to 10% SDS-PAGE under reducing conditions (29). Proteins in the gel were electrophoretically transferred onto PVDF membranes, incubated overnight at 4 C with 4 µg/mL immunopurified rabbit IgG directed against human glycodelin (30), and visualized with Enhanced ChemiLuminescence reagents (ECL, Dupont, Wilmington, DE), according to the manufacturer’s instructions.

Gene constructions and glycodelin gene promoter analyses

Vectors expressing the rabbit PR (pKSV-rPR) were generated by inserting the entire open-reading frame of the rabbit PR complementary DNA into the BglII site of pKSV10 (Pharmacia, Uppsala, Sweden), as described (31). Plasmids expressing the human PRB (pGS5-hPR) were obtained by ligating the entire coding region of human PR into the EcoRI site of pGS5 (Stratagene, La Jolla, CA) (32). The PRE2-TATA-CAT reporter construct, containing two canonical PREs and a TATA-box cloned immediately upstream from the chloramphenicol acetyl transferase complementary DNA, was prepared as described (32). Transcriptional regulation of the human glycodelin promoter was evaluated using transient DNA transfection analyses. HeLa cells (American Type Cell Collection, Bethesda, Maryland) were cultured in DMEM supplemented with 10% FCS, L-glutamine, ampicillin, and gentamicin. Ishikawa cells (a well-differentiated endometrial adenocarcinoma cell line obtained from the University of California, San Francisco Cell Culture Facility) were cultured in the same medium with 25 mmol/L HEPES and 10 nmol/L estradiol added. Plasmid DNA was transfected into the cells using the calcium phosphate coprecipitation method. Reporter vectors were constructed using a 1120-base Hind III-SacI fragment extending 1100 bases upstream from the unique transcriptional start site on the human glycodelin (PP14) gene (12), subcloned into the XbaI site of pCAT (Promega Corp., Madison WI). Subconfluent cells (~250,000), in 10-cm culture plates, were transfected with 2 µg of the -1100 CAT reporter construct and 5 µg of an expression vector encoding the rabbit, human, or chicken PR genes. All experiments were standardized to 20 µg DNA using herring sperm DNA. After 24 h, the culture media were replaced with media containing 10% charcoal-treated calf serum, L-glutamine, ampicillin, gentamicin, and steroid hormones or antihormones in a final concentration of 0.1% ethanol. Control experiments received an equivalent amount of ethanol as vehicle. The cells were cultured in the presence of hormones or antihormones for 24 h, after which the cells were scraped and sonicated as described (33).

CAT reporter activity was determined by incubating the cell sonicates with [14C]chloramphenicol, which were extracted with ethyl acetate and subjected to silica gel thin-layer chromatography. The migration of acetylated [14C]chloramphenicol substrate was determined by autoradiography and quantified by excision and direct scintillation counting of the silica gel. The acetylated [14C]chloramphenicol counts were normalized to cell number, as described above.

Statistical analyses

Each experiment was performed a minimum of three times. The results are expressed as mean ± SE of n independent experiments. Because of the small sample sizes, differences among treatment groups were compared using nonparametric Kruskal-Wallis ANOVA. Dose-response experiments were analyzed by two-factor ANOVA with repeated measures. Two-tailed tests with P < 0.05 were accepted as significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Expression and immunolocalization of glycodelin in human endometrium

Purified rabbit antihuman glycodelin IgG was used to immunostain paraformaldehyde-fixed sections of human endometrium from ovulatory women. As has been reported previously, glycodelin was not detected in proliferative-phase endometrium (Fig. 1AGo), but was abundantly expressed in the glandular epithelial cells, but not in the stroma of midsecretory-phase endometrium (Fig. 1BGo). Control sections stained with nonimmune rabbit IgG showed no specific reaction product (data not shown).



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  		Figure 1. Paraformaldehyde fixed, paraffin-imbedded biopsies of proliferative (day 10, A) and midsecretory (day 22, B) human endometrium were stained with antiglycodelin antiserum using the immunoperoxidase method. Glycodelin was detected only in secretory glandular epithelium (B).

 
In vitro expression of glycodelin in purified primary human endometrial cell cultures

Highly purified human endometrial epithelial and stromal cells were cultured from midsecretory-phase endometrium (27). Metabolic labeling of secreted proteins was accomplished by incubating the cultures for 18–24 h in the presence of [35S]methionine and [35S]cysteine. Conditioned media from matched stromal and epithelial cell cultures were separated by SDS-PAGE and subjected to fluorography, which demonstrated a prominent 28-kDa band in the epithelial cell-conditioned media that was not present in conditioned media from stromal cells isolated from the same endometrial biopsies (Fig. 2AGo). Western blotting of conditioned media, using the rabbit antihuman glycodelin IgG, revealed that the 28-kDa band in epithelial cell supernatants was glycodelin (Fig. 2BGo). Metabolic labeling and Western blotting of epithelial and stromal cells, obtained from proliferative-phase endometrium, were negative for glycodelin expression. Likewise, Ishikawa adenocarcinoma cell-conditioned media failed to demonstrate glycodelin secretion, either in the presence or absence of added progestins, as reported previously (34).



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  		Figure 2. Stromal and epithelial cells were isolated and purified from a secretory-phase endometrial biopsy. Confluent wells of pure cells were incubated in methionine-free medium containing 2.5% FBS for 4 h; 50 µCi [35S]methionine and [35S]cysteine were added for 18 h. Secreted, de novo synthesized proteins were separated in 10% polyacrylamide gels and were fluorographed. A major band, at 28 kDa, was observed in the epithelial cell (epi) but not stromal cell (strom) supernatants (A). Western blotting of unlabeled epi supernatants verified that this band corresponds to glycodelin (B).

 
Regulation of glycodelin secretion from human endometrial epithelial cells by progestins and antiprogestins

To investigate the possible regulation of glycodelin by progestins, we used purified endometrial epithelial cells collected from proliferative-phase biopsies. As described above, and unlike epithelial cells derived from secretory endometrium, epithelial cells from proliferative endometrium did not secrete glycodelin under basal culture conditions. However, incubation of these epithelial cells with increasing concentrations of R5020 resulted in a progressive increase in glycodelin secretion, with a half-maximal stimulation of CAT activity (EC50) = 0.5 nmol/L (Fig. 3AGo). This concentration correlates well with the dissociation constant of the human endometrial PR (35). Further metabolic labeling experiments were performed using the progestin R5020 (n = 5) and the antiprogestin RU486 (n = 4) at a concentration of 10 nmol/L. Both agents had significant agonistic activity on glycodelin secretion in isolated epithelial cells, stimulating glycodelin secretion 7.5 ± 2.6-fold and 6.6 ± 3.9-fold, respectively, over untreated epithelial cells (P = 0.04, Kruskal-Wallis test, Fig. 3BGo).



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  		Figure 3. A, Purified epithelial cells isolated from a proliferative-phase endometrial biopsy were established as described in Fig. 2Go. Replicate, confluent wells were incubated for 24 h, in the presence of 50 µCi [35S]methionine and [35S]cysteine, without or with increasing concentrations (10-11–10-8 mol/L) of the synthetic progestin R5020. A dose-response effect of the added progestin was observed on the intensity of 28-kDa glycodelin synthesized and secreted into the cell culture supernatants. B, Quantification of glycodelin, detected after metabolic labeling of nascent, secreted proteins from endometrial epithelial cells cultured without added hormone or in the presence of 10 nmol/L R5020 (n = 5) or 10 nmol/L RU486 (n = 4). Fluorograms of gels from cultures prepared as described in Fig. 3AGo were captured and digitized using BioImage software. The data were normalized to cellular DNA content of each well; and the means (±SE) were expressed, relative to the untreated control wells for each independent cell preparation (n).

 
Transcriptional regulation of the glycodelin gene promoter in HeLa cells

Cotransfection of the -1100 glycodelin promoter CAT construct with vectors expressing the rabbit PR resulted in hormone-induced transcription and expression of the CAT reporter gene. CAT activity in the cotransfected cells demonstrated a dose-response effect to R5020 (Fig. 4AGo). The effects of R5020 and RU486 were studied, over a wide range of hormone concentrations, in multiple experiments. As observed at the protein level in primary endometrial epithelial cells, RU486 acted as an agonist on the glycodelin gene promoter in transfected HeLa cells. The mean results (±SE) of replicate dose-response curves are shown in Fig. 4BGo. The responses to R5020 and RU486 were essentially superimposable and not statistically different (two-factor ANOVA with repeated measures, P = 0.74), with maximum activation of CAT between 1–10 nmol/L for both ligands. The EC50 for both R5020 and RU486 was 0.5 nmol/L. Progesterone and ZK299 (onapristone) also were agonists on the -1100 glycodelin promoter, displaying EC50 concentrations of 1 nmol/L and 10 nmol/L, respectively, whereas estradiol (1 nmol/L and 10 nmol/L) had no effect on CAT activity (data not shown). Identical results were observed when glycodelin promoter contructs were transfected into Ishikawa adenocarcinoma cells (Vigne and Taylor, unpublished results).



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  		Figure 4. A, HeLa cells were cotransfected with a pCAT construct (Promega Corp.) containing the 1100 bp human glycodelin gene promoter and a rabbit progesterone receptor expression vector. The cells were stimulated for 24 h without or with increasing concentrations of R5020 (10-11–10-8 mol/L). Cell extracts were prepared, and CAT activity was quantified using [14C]chloramphenicol and thin-layer chromatography. B, A series of R5020 and RU486 dose-response experiments were carried out as described in Fig. 4AGo. The means (±SE) of 2–5 independent CAT assays demonstrated that progestin concentrations comparable with physiological progesterone levels and similar concentrations of RU486 induced a 4- to 9-fold increase in glycodelin gene promoter activity. The effects of the two ligands were not statistically different, by two-factor ANOVA with repeated measures (P = 0.74).

 
We compared the regulation of the -1100 glycodelin gene promoter with that of a classical, minimal promoter containing two adjacent consensus PRE elements [PRE2TATA-CAT (33)]. Cotransfection of PR with PRE2TATA-CAT, in the presence of 1 nmol/L R5020, increased CAT activity 4-fold, whereas the combination of 1 nmol/L R5020 plus 1 nmol/L RU486 caused a 40% inhibition of the induction of PRE2TATA-CAT activity by the pure progestin agonist (R5020) alone (Fig. 5AGo). Thus, in the context of a classical promoter, RU486 functioned as a progestin antagonist. By contrast, in cotransfections of PR with the -1100 glycodelin promoter CAT construct, 1 nmol/L R5020 and 1 nmol/L RU486 independently stimulated CAT activity 7- and 9-fold, respectively, over controls; and the combination of R5020 plus RU486 showed a agonistic stimulation of 12-fold. The antiprogestin ZK299, at a concentration of 10 nmol/L, also had agonistic activity on this promoter (Fig. 5BGo). When transfections were performed in the absence of cotransfected rabbit PR, incubation with 1 nmol/L R5020 or RU486 failed to activate the -1100 glycodelin promoter, verifying the PR dependence of this activation (Fig. 5CGo).



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  		Figure 5. A, Cotransfection of PR with PRE2TATA-CAT reporter constructs in the presence of 1 nmol/L R5020 caused a 4-fold increase in CAT activity, whereas 1 nmol/L RU486 had no effect on basal (control) activity. The combination of 1 nmol/L R5020 plus 1 nmol/L RU486 caused a 40% inhibition of induction of PRE2TATA-CAT activity by the pure progestin agonist (R5020) alone. B, By contrast, in cotransfections of PR with the -1100 glycodelin promoter CAT construct, both 1 nmol/L R5020 and 1 nmol/L RU486 independently stimulated CAT activity 7- and 9-fold over controls. The antiprogestin ZK299, at a concentration of 10 nmol/L, also had agonistic activity on this promoter, and the combination of R5020 plus RU486, likewise, showed an agonistic stimulatory effect. C, Receptor dependence of the progestin and antiprogestin-induced activation of the -1100 glycodelin promoter was verified by cotransfections with (+rPR) and without (no PR) rabbit PR, followed by incubation with 1 nmol/L R5020 or 1 nmol/L RU486.

 
The species specificity of PR-induced CAT activation was evaluated. Cotransfection of human PRB with the -1100 glycodelin promoter reporter had effects identical to those observed with the rabbit PR. Both R5020 and RU486 stimulated CAT activity 5- to 6-fold (Fig. 6Go). Cells that were cotransfected with chicken PR and -1100 glycodelin promoter CAT vectors were stimulated by R5020, but these cells did not respond to RU486.



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  		Figure 6. Cotransfection of human PR with the -1100 glycodelin promoter reporter had effects identical to those observed with the rabbit PR (see Fig. 5CGo). Both R5020 and RU486 stimulated CAT activity 5- to 6-fold. Cells that were cotransfected with chick PR and -1100 glycodelin promoter CAT vectors were stimulated by R5020, but these cells did not respond to RU486, which has been shown previously not to bind to the chick PR.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In cycling human endometrium, glycodelin expression was confined to the epithelial glands of secretory-phase tissue, as reported previously (18, 19). In freshly isolated human endometrial epithelial and stromal cells, only the epithelial cells derived from secretory-phase biopsies secreted glycodelin protein. However, in purified primary epithelial cell cultures derived from proliferative-phase biopsies, R5020 induced a dose-dependent increase in the de novo synthesis and secretion of glycodelin in vitro. The EC50 of this response is consistent with the dissociation constant of the human PR. Surprisingly, we observed that the antiprogestin, RU486, also had agonistic activity on glycodelin secretion by proliferative-phase endometrial epithelial cells.

This observation was corroborated in transient transfection assays using two human müllerian-derived epithelioid cell lines, HeLa and Ishikawa. Hormonal stimulation of cells cotransfected with rabbit PR and the -1100 glycodelin promoter CAT construct resulted in a 4- to 9-fold stimulation of CAT activity. The same approximate magnitudes of activation were observed using progestin agonists (R5020, progesterone) and antagonists (RU486, ZK299). By contrast, in HeLa cells cotransfected with PR and the consensus PRE2TATA-CAT vector, R5020 and progesterone behaved as agonists, whereas RU486 had classical antagonist activity. A further control was afforded by cotransfecting HeLa cells with chicken PR and -1100 glycodelin promoter CAT vectors. R5020 had agonist activity, but RU486 had no activity. The latter result is explained by the fact that RU486 fails to bind the chicken PR (36). This finding, along with the negative controls shown in Fig. 5CGo, verify that the agonistic effect of RU486 on the -1100 glycodelin CAT reporter is mediated via PRs that have the capacity to bind this ligand.

Cotransfection of HeLa (Fig. 6Go) and Ishikawa (data not shown) cells with glycodelin promoter and human PRB expression vectors demonstrated agonist activity of R5020 and RU486. Other examples of agonist or partial agonist activity of RU486 have been reported previously (37, 38). In the study by Bamberger et al. (39), RU486 and medroxyprogesterone acetate (a synthetic C21 progestin) both were found to be agonists on the interleukin-2 antigen receptor response element (ARRE-1) gene promoter. The degree of agonistic activity of antiprogestin compounds seems to be controlled by the ratio of coactivators to corepressors recruited to the transcription complex by promoter-bound progestin receptors (40).

Analysis of the -1100 gene promoter sequence of human glycodelin reveals three consensus PRE half-sites at -1071, -746, and -304 bases from the transcription start site. We postulate that endogenous PR complexes present in primary human endometrial epithelial cells, or mammalian PRs cotransfected into HeLa and Ishikawa cells, bind ligand, translocate to these half-sites, and activate transcription of the glycodelin gene, as has been suggested by Welte et al. (41) for the glucocorticoid receptor regulation of casein gene transcription. Mutational analyses of the glycodelin gene promoter should allow us to test this hypothesis.

Our in vitro findings in isolated, purified human epithelial cells are in contrast to the apparent pharmacological effects of antiprogestins administered to women in vivo. The administration of onapristone to ovulatory, cycling women is reported to decrease the circulating concentrations of plasma glycodelin (42) and immunodetectable glycodelin expression in endometrial tissue (43). Whether these are direct effects of the antiprogestin on glycodelin synthesis or indirect effects caused by inhibition of endogenous progesterone production (44) is not known. The in vivo effects of RU486 have not been studied extensively. Weakly agonistic effects have been reported in the endometrium (45) and pituitary (46), particularly in the absence of endogenous progesterone. Other possible explanations for this discrepancy include disparate effects of glycodelin secretion in vivo and in vitro, or modulatory paracrine effects of endometrial stromal or immune cells (present in vivo but not in our in vitro models) on glycodelin gene expression and protein secretion. Future studies using coculture systems may clarify the nuances of glycodelin synthesis in endometrial epithelial cells.


   Acknowledgments
 
The helpful advice and assistance of Dr. Hugues Loosfelt, Dr. Synthia Mellon, Christophe Pichon, and Victor Chao are gratefully appreciated.


   Footnotes
 
1 These studies were supported by funds from the Philippe Foundation (to R.N.T.) and the Helsinki University Central Hospital Research Fund (to M.S.). Back

Received April 6, 1998.

Revised June 12, 1998.

Accepted July 17, 1998.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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