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Original Studies |
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.
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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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 
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.
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Subjects and Methods |
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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-(1-propynyl)-estra-4,9-dien-3-one)
were gifts from Dr. D. Philibert (Roussel-UCLAF, Romainville, France).
Onapristone (ZK299,
11ß-(4-dimethylaminophenyl)-17-hydroxy-17ß-(3-hydroxypropyl)-13-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-
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- 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.
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Results |
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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. 1A
), but was
abundantly expressed in the glandular epithelial cells, but not in the
stroma of midsecretory-phase endometrium (Fig. 1B). Control sections
stained with nonimmune rabbit IgG showed no specific reaction product
(data not shown).
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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. 2A). Western
blotting of conditioned media, using the rabbit antihuman glycodelin
IgG, revealed that the 28-kDa band in epithelial cell supernatants was
glycodelin (Fig. 2B). 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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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. 3A). 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. 3B).
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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. 4A). 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. 4B. 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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). 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. 5B). 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. 5C).
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). 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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Discussion |
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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. 5C
, 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. 6) 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.
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Acknowledgments |
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Footnotes |
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Received April 6, 1998.
Revised June 12, 1998.
Accepted July 17, 1998.
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References |
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1- and 2-PEG) and the
soluble placental proteins 12 and 14 (PP12 and PP14). Placenta. 7:283–294.[CrossRef][Medline]
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