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The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 9 3364-3377
Copyright © 1999 by The Endocrine Society

Original Studies

Treatment of Human Endometrial Gland Epithelial Cells with Chorionic Gonadotropin/Luteinizing Hormone Increases the Expression of the Cyclooxygenase-2 Gene

X.-L. Zhou, Z. M. Lei and Ch. V. Rao

Division of Basic Science Research, Department of Obstetrics and Gynecology, University of Louisville Health Sciences Center, Louisville, Kentucky 40292

Address all correspondence and requests for reprints to: Dr. Ch. V. Rao, Department of Obstetrics and Gynecology, 438 MDR Building, University of Louisville Health Sciences Center, Louisville, Kentucky 40292. E-mail: cvrao001{at}gwise.louisville.edu Web site:


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Endometrial glands contain higher levels of LH/hCG receptors than other cells in the human uterus. The present study investigated their functional importance. Northern and Western blotting and covalent receptor cross-linking demonstrated that human endometrial gland epithelial cells in culture contained multiple LH/hCG receptor transcripts and an 80-kDa receptor protein that can bind [125I]hCG in a hormone-specific manner. Culturing cells with highly purified hCG resulted in a time- and dose-dependent increase in steady state levels of cyclooxygenase-2 (COX-2) messenger ribonucleic acid and protein and the secretion of PGE2. Although human LH could mimic hCG, FSH, TSH, and {alpha}- or ß-subunits of hCG had no effect on COX-2 protein levels.

Studies on signaling revealed that treatment of cells with hCG resulted in an increase in cAMP levels and protein kinase A (PKA) activity. Inhibition of PKA activity by cotreatment with isoquinolinesulfonamide (H-89) prevented hCG from increasing COX-2 protein levels. Treatment with 8-bromo-cAMP mimicked the effect of hCG, and cotreatment with a selective inhibitor of type I PKA, 8-chloro-cAMP, prevented 8-bromo-cAMP and hCG from increasing COX-2 protein levels.

The requirement of receptors for LH/hCG action was investigated by 24-h treatment of human endometrial gland epithelial cells with 21-mer phosphorothioate oligodeoxynucleotides (ODNs) synthesized from human receptor sequence. Treatment with 2 µmol/L antisense, but not sense, ODN resulted in a dramatic reduction in LH/hCG receptor protein levels. hCG was unable to increase COX-2 protein, PGE2, and cAMP levels in an antisense, but not in sense, ODN-treated cells.

In summary, we conclude that hCG and LH treatment can increase expression of the COX-2 gene in human endometrial gland epithelial cells. The effect was time and dose dependent, hormone specific, and mediated by the cAMP/type I protein kinase A signaling pathway. The hCG actions require a normal complement of its receptors in cells. These hCG and LH effects may be another action of these hormones in human endometrium that is important for implantation of the blastocyst and continuation of pregnancy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT WAS NOT until about 9 yr ago that we realized the human uterus could be directly regulated by LH/hCG. This possibility first arose from findings that the human uterus immunostains for LH/hCG receptors (1). Subsequent studies revealed that the human uterus contains LH/hCG receptor transcripts and receptor protein that can bind [125I]hCG (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). The uterine receptors are functional. For example, LH and hCG treatment can increase vasodilatory and decrease vasoconstrictive eicosanoids and can decrease resistance index in human uterine arteries (9, 13). These actions are consistent with hCG playing a major role in a dramatic increase in uterine blood flow that normally occurs during pregnancy. LH and hCG can stimulate cell proliferation and inhibit contractions through down-regulation of gap junctions and inhibition of intracellular Ca2+ levels in human myometrial smooth muscle cells (6, 14, 15, 16). These actions are consistent with the role of hCG in enlarging myometrium during pregnancy and keeping it quiescent until labor begins (6, 8, 14, 15). LH and hCG can also promote morphological as well as functional differentiation of endometrial stromal cells into decidua (17, 18). This effect is mediated by increasing PGE2 synthesis through up-regulating the expression of the COX-2 gene via a posttranscriptional mechanism and also by increasing cAMP levels (17, 19, 20). The hCG effect on functional differentiation, as measured by PRL production, involves an increase in the transcription rate of the prolactin gene rather than a decrease in the degradation of its transcripts (18). These actions in human endometrial stromal cells are consistent with the role of LH/hCG in promoting implantation of blastocyst and maintenance of pregnancy. Structural and functional changes in endometrial glands may also be important for the implantation process. The glands contain higher LH/hCG receptor levels than myometrial and endometrial stromal cells and uterine arteries (1). The higher receptor levels coupled with an increase during the luteal phase suggest that LH/hCG actions in endometrial gland epithelial cells may also be relevant to implantation. COX-2, which catalyzes the formation of eicosanoids, is absolutely essential for the implantation process (21). This enzyme in human endometrial stromal cells and human fallopian tube epithelial cells is responsive to hCG stimulation (19, 22). Because of the possible relevance of LH/hCG actions to implantation through its actions on endometrial glands (22, 23), we focused our studies on COX-2. The results demonstrate that hCG and LH can indeed increase expression of the COX-2 gene via cAMP/type I protein kinase A signaling pathway, and hCG cannot increase COX-2 in the absence of a normal complement of its receptors in the cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

The following items were obtained as gifts: polyclonal anti-LH/hCG receptor antibody raised against a synthetic N-terminus amino acid sequence of 15–38 from Dr. Patrick Roche at the Mayo Clinic (Rochester, MN); LH/hCG receptor complementary DNA (cDNA) from Dr. Hugues Loosfelt at Hospital de Bicetre (Paris, France); cDNA of COX-2 from Dr. Harvey Herschman at the University of California at Los Angeles; cDNA of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Dr. Russell Prough of our institution; highly purified hCG (CR-127; 14,900 IU/mg), human LH (AFP-0264B; 4,015 IU/mg), human FSH (AFP-87929B; 1,683 IU/mg), human TSH (AFP-4314C; 15 IU/mg), {alpha}- (CR-125) and ß- (CR-125; 29 IU/mg) subunits of hCG from the National Hormone and Pituitary Program supported by the NIDDK, NICHHD, and USDA (Rockville, MD); and 8-chloro (cl)-cAMP from Drug Synthesis and Chemistry Branch, NCI (Bethesda, MD). The following items were purchased: polyclonal anti-COX-2 antibody from Oxford Biochemical Research (Oxford, MI); enzyme immunoassay kits for PGE2 and cAMP from Cayman Chemical Co. (Ann Arbor, MI); horseradish peroxidase-labeled antirabbit IgG, antibiotic-antimycotic solution, FBS, trypsin, and insulin from Life Technologies, Inc. (Grand Island, NY); in vitro transcription and random prime labeling kits, pre-Tag nonradioactive protein kinase A (PKA) and protein kinase C (PKC) activity measurement kits from Promega Corp. (Madison, WI); Nonidet P-40, Percoll, 10 x HBSS, DMEM-Ham’s F-12 nutrient mixture (DMEM/F-12 medium), phenylmethylsulfonylfluoride (PMSF), aprotinin, leupeptin, collagenase (type IA-S; 320 U/mg solid), monoclonal anti-ß-tubulin antibody, and 8-bromo-cAMP (8-Br-cAMP) from Sigma Chemical Co. (St. Louis, MO); sodium azide from American Bioanalytical Co. (Natick, MA); enhanced chemiluminescence Western blot detection kit from Amersham Pharmacia Biotech (Arlington Heights, IL); BS3 [bis-(sulfosuccimidy)suberate] from Pierce Chemical Co. (Rockford, IL); [{alpha}-32P]CTP and [{alpha}-32P]deoxy-CTP (800 Ci/mmol) from DuPont-New England Nuclear Corp. (Boston, MA); the monoclonal antivimentin, anticytokeratin, anti-{alpha} smooth muscle actin and anti-CD68 antibodies from DAKO Corp. (Carpenteria, CA); bisindolylmaleimide (Bis) and isoquinolinesulfonamide (H-89) from Calbiochem (San Diego. CA); and peroxidase substrate and avidin-biotin immunoperoxidase kits from Vector Laboratories, Inc. (Burlingame, CA). The 21-mer phosphorothioate antisense (5'-GCCGAGAACCGCTGCTTCATG-3') and sense (5'-CATGAAGCAGCGGTTCTCGGC-3') oligodeoxynucleotides (ODNs) were synthesized from human LH/hCG receptor cDNA sequence beginning at the ATG translation initiation codon.

Collection of tissues

Endometrial specimens were obtained from 22- to 45-yr-old premenopausal women undergoing hysterectomy or diagnostic dilatation and curettage for a variety of medical indications other than endometrial cancer. Only those found to be histologically normal and dated to be in the proliferative or secretory phase were used in the studies. The use of these tissues was approved by our institutional human studies committee. The tissues were immediately brought to the laboratory on ice in sterile tubes containing DMEM/F-12 medium, pH 7.4, with 10% FBS and 1% antibiotic-antimycotic mixture (10,000 U penicillin G sodium, 10 mg/mL streptomycin sulfate, and 25 µg/mL amphotericin and fungizone in 0.85% saline).

Isolation and culture of cells

Endometrial gland epithelial cells were isolated and cultured with few modifications from previously described procedures (25, 26). Briefly, endometrial tissues were cut into 1-mm3 pieces and digested with 0.25% collagenase in DMEM/F-12 medium in capped sterile tubes in a shaking water bath for 30 min at 37 C. The digest was then passed first through a 250-µm pore size nylon mesh and then through a 35-µm pore size nylon mesh. The glandular epithelial cells retained on the mesh were backwashed with 20 mL 1 x HBSS containing 1% antibiotic-antimycotic mixture. The backwash solution containing enriched glandular epithelial cells was centrifuged for 5 min at 200 x g. The cell pellet was resuspended in DMEM/F-12 medium containing 10% FBS and incubated for 1 h at 37 C in an incubator containing a humidified atmosphere of 5% CO2-95% air. Unattached cells were discarded, and the purity of the attached cells was determined by immunocytochemistry using anticytokeratin (epithelial), antivimentin (stromal), anti-{alpha} smooth muscle actin, and anti-CD68 (macrophages) antibodies. Cell viability, which was greater than 95%, was determined by trypan blue exclusion. After reaching subconfluence in about 4–6 days, the medium was replaced with fresh DMEM/F-12 containing no FBS and the indicated concentrations of hCG or other hormones. The number of cells used per culture well or flask varied among the experiments. However, the same number of cells was used within the same experiments.

Northern blotting

For this procedure, total ribonucleic acid (RNA) was isolated from endometrial gland epithelial cells, and then 30-µg aliquots were heat denatured at 100 C for 5 min, resolved on formaldehyde-agarose gels, and blotted onto Gene Screen Plus membranes (27). The RNA was cross-linked to the membranes by irradiation for 2.5 min under UV light and baking for 10 min at 70 C. Nonspecific binding was blocked by salmon sperm DNA, and the blots were sequentially hybridized overnight at 65 C with either 1–5 x 107 cpm/mL 32P-labeled LH/hCG receptor or 0.5–1 x 107 cpm/mL 32P-labeled COX-2 riboprobes transcribed from corresponding cDNAs by an in vitro transcription kit. Then the membranes were washed at 65 C twice with 2 x SSC (1 x SSC = 150 mmol/L sodium chloride and 15 mmol/L sodium citrate, pH 7.4) containing 0.1% SDS and, again, twice with 0.1 x SSC-0.1% SDS. The washed membranes were exposed for 48–72 h at -70 C to Kodak XAR-5 film (Eastman Kodak Co., Rochester, NY) with intensifying screens. The membranes were stripped and rehybridized at 42 C with 2–5 x 106 cpm/mL 32P-labeled GAPDH cDNA prepared by using a random priming kit. The membranes were washed as described above at 42 C. The molecular size of the transcripts was determined by running a RNA ladder in an adjacent lane.

Western blotting

For this procedure, endometrial gland epithelial cells were homogenized in buffer containing 200 mmol/L PMSF and 20 mmol/L leupeptin to inhibit the activity of endogenous proteases. Then, 30-µg aliquots of protein were separated by discontinuous 10% SDS-PAGE under reducing conditions (28). The separated proteins were electroblotted onto Immobilon-P (Millipore Corp., Bedford, MA) membranes (29). After blocking nonspecific binding sites with 5% nonfat dry milk in 5 mmol/L Tris-HCl (pH 7.4), 136 mmol/L NaCl, and 0.1% Tween-20 (TBST buffer), the blots were incubated with a 1:1500 dilution of anti-LH/hCG receptor antibody or a 1:1000 dilution of anti-COX-2 antibody for 2 h at 22 C, washed twice for 10 min each time with TBST buffer, and again washed once with TBST containing 40 mmol/L sodium azide. The washed blots were reincubated for 1 h at 22 C with a 1:2000 dilution of horseradish peroxidase-labeled antirabbit IgG and washed as described above. Then, the binding of anti-LH/hCG receptor and anti-COX-2 antibodies was detected by an enhanced chemiluminescence Western blotting detection kit. To correct the differences in protein loading, after the detection of COX-2 protein, membranes were washed with TBST buffer containing sodium azide for 30 min, then incubated with a 1:2000 dilution of anti-ß-tubulin antibody and washed as before. The molecular sizes of the LH/hCG receptor and COX-2 proteins were determined by running molecular size marker proteins in an adjacent lane. In the procedural control, receptor antibody was preabsorbed with excess LH/hCG receptor peptide, and the primary antibody was omitted in case of COX-2.

Covalent receptor cross-linking

For this procedure, 100-µg aliquots of homogenate protein were incubated for 30 min at 37 C with 1–2 x 106 cpm/mL [125I]hCG in the presence or absence of 5 µg unlabeled hCG (30). The hCG was radioiodinated by a lactoperoxidase technique to a specific activity of 76.1 µCi/µg (31). The receptor-bound [125I]hCG was cross-linked by incubating for 1 h at room temperature with 100 mmol/L BS3 dissolved in 5 mmol/L sodium citrate buffer, pH 5.0. Then the reaction was stopped by the addition of 50 mmol/L Tris-HCl, pH 7.5, and centrifugation for 30 min at 27,000 x g at 4 C. Pellets containing [125I]hCG-receptor complexes were solublized with 1% Triton X-100; diluted with 125 mmol/L Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, and 10% mercaptoethanol; and separated on 8% SDS-PAGE under nonreducing conditions. The gel was fixed, dried, and exposed for 1–3 days to Kodak XAR-5 film with intensify screens at -70 C. Molecular sizes of the [125I]hCG receptor complexes and unbound [125I]hCG were determined by running mol wt marker proteins in an adjacent lane.

Measurement of medium PGE2 levels

PGE2 levels in duplicate 50-µL medium samples were quantified using a commercial kit. Instructions provided in the kits were followed. The specificity of the PGE2 antibody was 100% for PGE2, 43% for PGE3, 18.7% for PGE1, and 1% or less for all other eicosanoids tested. Intra- and interassay coefficients of variation were less than 10%. The detection limit of the assay was 29 pg/mL PGE2.

Measurement of medium cAMP levels

Levels of cAMP in 50-µL aliquots of medium samples were quantified using a commercial kit. Instructions provided in the kit were followed. The specificity of the cAMP antibody was 100% for acetylated cAMP, 0.3% for cAMP, 0.05% for acetylated cGMP, and 0.01% or less for cGMP, acetylated adenosine, cytidine, guanoisine, and uridine. Intra- and interassay coefficients of variation were less than 10%. The detection limit of the assay was 1.1 pmol/mL.

Immunocytochemistry

The cells were fixed in Bouin’s solution for 30 min and then immunostained by an avidin-biotin immunoperoxiadase method using a 1:500 dilution of anti-LH/hCG receptor, anticytokeratin, antivimentin, anti-{alpha} smooth muscle actin, and anti-CD68 antibodies (1). For the procedural controls, the primary antibodies were replaced with nonspecific IgG.

Measurement of PKA and PKC activities

Cells were lysed by sonication and freezing/thawing in 200 µL 25 mmol/L Tris-HCl buffer, pH 7.5, containing 1 mmol/L ethylenediamine tetraacetate, 1 mmol/L dithiothreitol, 20 mmol/L NaCl, 0.5 mmol/L PMSF, 1 µmol/L aprotinin, and 50 µmol/L leupeptin. PK activities were determined by incubating 3–5 µg lysate protein for 30 min at 30 C with fluorescent-labeled A1 peptide for the PKA assay and fluorescent-labeled C1 peptide for the PKC assay. Nonphosphorylated and phosphorylated fluorescent peptides were separated on 0.8% agarose gels. Phosphorylated fluorescent peptide bands were excised and eluted, and the optical density at 570 nm was measured using a 96-well plate reader. PK activities were calculated from the densitometric values using instructions provided by the kit manufacturer. Positive and negative controls supplied in the kits were assayed at the same time with endometrial cell samples.

Culturing cells with antisense and sense ODNs

Cells were cultured in six-well plates (5 x 105 cells/well) for 24 h in serum- and phenol red-free medium containing 2 µmol/L antisense or sense ODNs. After 24 h, immunocytochemistry for LH/hCG receptors was performed on some cells, whereas others were cultured for 4 h in the presence or absence of 100 ng/mL hCG. Media were then removed for the measurement of PGE2 and cAMP levels, and cells were recovered for the measurement of COX-2 protein by Western blotting.

Densitometry

Optical densities of autoradiographic bands of COX-2 and GAPDH messenger RNAs (mRNAs) and COX-2 and ß-tubulin protein bands were measured in a linear range using a Z-gel scanning system (Zaxis, Hudson, OH).

Replication of experiments and statistical analyses

Each experiment was duplicated and repeated at least three times on cells from different endometrial specimens. Data from all of the experiments were pooled for calculation of the means and SE and for one-way ANOVA and Duncan’s multiple range test (32).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Purity of human endometrial gland epithelial cells in culture

Purity was determined by immunocytochemistry using four different antibodies, three of which are directed against common cell contaminants. As shown in Fig. 1Go, virtually all cells were immunostained with anticytokeratin antibody, which binds to epithelial cells (Fig. 1AGo). Antibodies that recognize fibroblasts (vimentin; Fig. 1BGo), smooth muscle cells (Fig. 1CGo), and macrophages (CD68; Fig. 1DGo) did not immunostain the cells. The procedural control showed no immunostaining (Fig 1EGo).



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  		Figure 1. Immunocytochemical demonstration of the purity of human endometrial gland epithelial cells in culture. The immunostainings for cytokeratin (A), vimentin (B), {alpha} smooth muscle actin (C), and CD68 (D) are shown. E is an immunostaining control in which nonspecific IgG was used. Magnification, x150.

 
LH/hCG receptors

Even though previous studies showed that glands in intact tissue contained higher LH/hCG receptor levels than the other uterine cells (1, 2, 3, 7), we needed to reestablish that endometrial gland epithelial cells in culture retained these receptors. Northern analysis demonstrated that cells contained a major 4.3-kb and minor 3.6-, 2.4-, and 1.8-kb transcripts of LH/hCG receptors (Fig. 2Go, lane 1). Western blotting showed that cells contained 80-kDa protein (lane 2), which was not detected when the receptor antibody was preabsorbed with the excess receptor peptide (lane 3). Covalent receptor cross-linking revealed that [125I]hCG forms a complex with a molecular size of 125 kDa (lane 4). Formation of this complex was inhibited when excess unlabeled hCG (lane 5) and LH (lane 6), but not when TSH (lane 7), FSH (lane 8), and {alpha}- (lane 9) and ß- (lane 10) subunits of hCG were present in the incubation medium. The 45-kDa band represents unbound [125I]hCG, and the 80-kDa difference between 125 and 45 kDa represents the molecular size of free receptor protein.



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  		Figure 2. Northern blotting (lane 1), Western blotting (lanes 2 and 3), and covalent receptor cross-linking (lanes 4–10) for LH/hCG receptors in human endometrial gland epithelial cells. Lane 3 is receptor antibody preabsorption control, and lanes 5, 6, 7, 8, 9, and 10 contain excess unlabeled hCG, LH, TSH, FSH, and {alpha}- and ß-subunits of hCG, respectively. 125 kDa represents the size of the [125I]hCG-receptor complex, and 45 kDa represents the size of unbound [125I]hCG. The 80-kDa difference represents the molecular size of receptor protein.

 
Effect of hCG on COX-2 expression

Endometrial gland epithelial cells contained an expected 4.4-kb transcript of COX-2 (Fig. 3Go). Transcript levels significantly increased in a time- and dose-dependent manner after treatment with highly purified hCG compared with control values. The hCG effect was specific because mRNA levels of a housekeeping gene, GAPDH, were unchanged by treatment.



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  		Figure 3. Time and dose dependency of the hCG effect on steady state COX-2 mRNA levels in human endometrial gland epithelial cells. In time dependency studies, cells were cultured with 100 ng/mL hCG for increasing lengths of time. Cells cultured in the absence of hCG for the same lengths of time served as controls. In dose dependency studies, cells were cultured for 4 h in the presence or absence (controls) of increasing concentrations of hCG. In this figure and all of the others, the mean and SE of values from at least three separate experiments performed in triplicate are presented. The inset shows a representative Northern blot. The COX-2/GAPDH densitometric ratios in controls were considered to be 100%. *, P < 0.05 compared with the controls.

 
Figure 4Go shows that endometrial gland epithelial cells contained 72-kDa COX-2 protein, and its levels also significantly increased in a time- and dose-dependent manner after treatment with hCG compared with control values. The increase in COX-2 protein was parallel to, but not identical with, an increase in COX-2 mRNA.



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  		Figure 4. Time and dose dependency of the hCG effect on COX-2 protein levels in human endometrial gland epithelial cells. In time dependency studies, cells were cultured with 100 ng/mL hCG for increasing lengths of time. Cells cultured in the absence of hCG for the same lengths of time served as controls. In dose dependency studies, cells were cultured for 4 h in the presence or absence (controls) of increasing concentrations of hCG. The insets show representative Western blot. The optical density of the COX-2 band in controls was considered 100%. *, P < 0.05 compared with the controls.

 
The hCG effect on COX-2 protein levels was mimicked by LH, but not by FSH and TSH, which are in the same glycoprotein hormone family, or by isolated {alpha}- and ß-subunits (Fig. 5Go). These data suggest that the hCG effect is hormone specific and requires the conformation of native hormone.



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  		Figure 5. Hormone specificity of the hCG effect on COX-2 protein levels. Human endometrial gland epithelial cells were cultured for 4 h in the presence or absence of various hormones. The inset shows a representative Western blot. The optical density of the COX-2 band in controls was considered to be 100%. *, P < 0.05 compared with the controls.

 
As an increase in COX-2 protein does not necessarily mean that the enzyme is catalytically active, we measured PGE2 levels in medium. Figure 6Go shows that hCG treatment resulted in a time- and dose-dependent increase in PGE2 levels in medium. The increase in PGE2 levels paralleled the increase in COX-2 protein.



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  		Figure 6. Time and dose dependency of the hCG effect on medium PGE2 levels in human endometrial gland epithelial cells. In time dependency studies, cells were cultured with 100 ng/mL hCG for increasing lengths of time. Cells cultured in the absence of hCG for the same lengths of time served as controls. In dose dependency studies, cells were cultured for 4 h in the presence or absence (control) of increasing concentrations of hCG. The PGE2 levels in the controls were considered to be 100%. *, P < 0.05 compared with the controls.

 
Signaling in hCG action

As cAMP is a common signaling molecule involved in hCG and LH actions, we first investigated the hCG effect on its levels in medium of endometrial gland epithelial cells. Figure 7Go shows that hCG treatment had a significant time- and dose-dependent stimulatory effect on cAMP levels. The first increase in cAMP levels preceded an increase in COX-2 mRNA and protein levels.



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  		Figure 7. Time and dose dependency of the hCG effect on medium cAMP levels in human endometrial gland epithelial cells. In time dependency studies, cells were cultured with 100 ng/mL hCG for increasing lengths of time. Cells cultured in the absence of hCG for the same lengths of time served as controls. In dose dependency studies, cells were cultured for 2 h in the presence or absence (control) of increasing concentrations of hCG. The cAMP levels in controls were considered to be 100%. *, P < 0.05 compared with the controls.

 
The increase in cAMP levels due to hCG treatment would suggest that PKA activity also may have been increased. Figure 8Go shows that hCG treatment did indeed significantly increase PKA activity compared with the control value. This effect was specific, because hCG treatment had no effect on PKC activity. These data indicate that PKA, but not PKC, activation is probably required for the hCG effect. If this is the case, then inhibition of PKA, but not PKC, activity should prevent hCG from increasing COX-2 protein levels. When this prediction was tested, we found that cotreatment with the PKA inhibitor, H-89, but not cotreatment with the PKC inhibitor, Bis, prevented an increase in COX-2 protein due to hCG treatment (Fig. 9Go). The inhibitors alone had no effect, suggesting that these signaling pathways do not control basal COX-2 protein levels.



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  		Figure 8. Treatment of human endometrial gland epithelial cells with hCG increases PKA, but not PKC, activity. Cells were cultured for 2 h in the presence or absence (control) of 100 ng/mL hCG. The inset shows a representative profile of phosphorylated and nonphosphorylated synthetic substrate peptides. *, P < 0.05 compared with the controls.

 


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  		Figure 9. Abrogation of the hCG effect on COX-2 protein levels in human endometrial gland epithelial cells by cotreatment with H89, a PKA inhibitor, but not with Bis, a PKC inhibitor. Cells were cultured for 4 h in the presence or absence (control) of hCG, Bis, or H-89 alone or their indicated combinations. The inset shows a representative Western blot. The COX-2/ß-tubulin densitometric ratio in controls was considered to be 100%. *, P < 0.05 compared with the control.

 
Although these data provide compelling reasons to believe that the cAMP/PKA signaling pathway was involved in hCG action, we tested the expectation that 8-Br-cAMP treatment mimics hCG action. The results presented in Fig. 10Go demonstrate that treatment with 0.1 mmol/L 8-Br-cAMP increased COX-2 protein levels just as did treatment with hCG.



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  		Figure 10. 8-Br-cAMP mimics hCG, and the type 1 PKA inhibitor, 8-cl-cAMP, prevents hCG and 8-Br-cAMP from increasing COX-2 protein levels. Cells were cultured for 4 h in the presence or absence (control) of hCG, 8-Br-cAMP, and 8-cl-cAMP alone or their indicated combinations. The inset shows a representative Western blot. The COX-2/ß-tubulin densitometric ratio in controls was considered to be 100%. *, P < 0.05 compared with the control.

 
There are two forms of PKA, and to determine which one was involved in hCG and 8-Br-cAMP actions, we used 8-cl-cAMP, a selective inhibitor of type 1 enzyme (33). The data in Fig. 10Go show that it prevented not only 8-Br-cAMP, but also hCG, from increasing COX-2 protein levels. 8-cl-AMP alone had no effect, suggesting that type I PKA activity does not control basal levels of COX-2 protein.

Requirement of LH/hCG receptors in hCG action

We used antisense ODN approach to determine whether receptors are required for hCG actions. Antisense and sense ODNs were synthesized from human LH/hCG receptor sequence. To determine their effectiveness in inhibiting LH/hCG receptor synthesis, human endometrial gland epithelial cells were treated for 24 h with 2 µmol/L ODNs, and then immunocytochemistry was performed. The results presented in Fig. 11Go demonstrate that untreated cells were immunostained for LH/hCG receptors, and this immunostaining was absent in procedural controls, indicating that it was specific. Treatment with antisense, but not sense, ODN resulted in a dramatic reduction in LH/hCG receptor immunostaining.



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  		Figure 11. Inhibition of LH/hCG receptor synthesis in human endometrial gland epithelial cells by treatment with antisense, but not with sense, LH/hCG receptor ODNs. Cells were cultured for 24 h in the presence or absence (control) of 2 µmol/L LH/hCG receptor ODNs and then immunostained for LH/hCG receptor protein. Nonspecific IgG was used in the procedural control. Magnification, x300.

 
We then used ODN-treated cells to determine the hCG effects. The results presented in Fig. 12Go demonstrate that hCG could not increase COX-2 protein, PGE2, or cAMP (Fig. 12Go) levels in antisense ODN-treated cells. Sense ODN-treated cells, which contained a normal complement of receptors, responded to hCG treatment in the same way as untreated cells by increases in COX-2 protein, PGE2, and cAMP levels (Fig. 12Go). Thus, these data clearly indicate that hCG cannot work in the absence of a normal complement of its receptors in cells.



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  		Figure 12. Abrogation of the hCG effect on COX-2 protein and medium levels of PGE2 and cAMP in human endometrial gland epithelial cells treated with antisense, but not with sense, LH/hCG receptor ODNs. Cells were cultured for 24 h in the presence or absence of 2 µmol/L LH/hCG receptor ODNs and then treated for 4 h with 100 ng/mL hCG. The COX-2/ß-tubulin densitometric ratio and PGE2 and cAMP levels in the controls (not treated with ODNs or hCG) were considered to be 100%. *, P < 0.05 compared with the controls.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Endometrial glands are specialized structures that are responsible for the synthesis and secretion of numerous molecules that are required for the implantation of the blastocyst. It is very important, for this reason, to know what hormones can control endometrial gland functions. Although it was generally believed that ovarian steroid hormones are responsible for this control, until 9 yr ago no one ever suspected that LH and hCG could also potentially control endometrial gland functions. The first clue for this possibility came from a study that demonstrated that the human uterus contains LH/hCG receptors, with the highest levels in endometrial glands (1, 2, 3, 7). Endometrial gland receptors were likely to be functional just as were those in myometrial smooth muscle cells (6, 14, 15, 16), endometrial stromal cells (12, 17, 18, 19), and uterine arteries (9, 13), but there were no data.

We used primary cultures of endometrial gland epithelial cells to determine the functional importance of LH/hCG receptors. These cells, like glands in intact uterus, contain LH/hCG receptors. Northern analysis detected multiple transcripts, which is a hallmark feature of the LH/hCG receptor gene transcription in other nongonadal target tissues just as in gonadal tissues (34). Western blotting detected an 80-kDa receptor protein. Covalent receptor cross-linking revealed that this protein can bind hCG and LH, but not other hormones in the same glycoprotein hormone family or the isolated {alpha}- and ß-subunits of hCG.

For several reasons, we chose COX-2 to determine the functional importance of LH/hCG receptors in endometrial gland epithelial cells. First and foremost was that LH/hCG can increase the expression of COX-2 in human endometrial stromal cells and fallopian tube epithelial cells (19, 22), which makes it likely that these hormones might do the same in endometrial gland epithelial cells. Second, human endometrial glands contain the highest levels of COX, although whether it is COX-1 and/or COX-2 has not been previously determined (35). Third, eicosanoids that are generated as a result of COX-2 activation regulate a number of endometrial functions that are related to implantation (36).

The results of the present study demonstrated that endometrial gland epithelial cells contain the expected size COX-2 mRNA transcripts and protein, and hCG treatment can increase their levels in a time- and dose-dependent manner. The hCG effect was hormone specific and requires the conformation of native hormone. COX-2 protein increased by hCG treatment was catalytically active, as determined by the response of PGE2 secretion. Although PGE2 was used as a measure of an enzyme activity, the catalytic activity of COX-2 can lead to the formation of other eicosanoids in this pathway (37). The measurement of all of these products was beyond the scope of our study. Moreover, PGE2 among these products has greater relevance to implantation.

The signaling involved in hCG action was first investigated by examining changes in cAMP levels in endometrial gland epithelial cells. The results demonstrated that hCG treatment can increase its levels in a time- and dose-dependent manner. As the increase in cAMP levels suggested that PKA might have been activated, we then measured PKA activity and found that hCG treatment can increase its activity, but not the activity of PKC. If PKA activation is necessary for hCG to increase COX-2, then inhibition of PKA, but not PKC, should prevent hCG from increasing COX-2 protein. The use of H-89 and Bis, PKA and PKC inhibitors, respectively, validated this expectation. If cAMP was a mediator of hCG action, then a cAMP analog, such as 8-Br-cAMP, should mimic hCG action, which it did.

Mammalian cells contain type I and type II forms of PKA (33). Studies with 8-cl-cAMP, a selective inhibitor of type I enzyme (33), revealed that it is the one that is involved in 8-Br cAMP and hCG actions. The extent of the type II enzyme’s contribution could not be determined, because there is no inhibitor of which we are aware for this enzyme.

As endometrial gland epithelial cells are relatively new targets of hCG/LH action, we investigated the requirement of receptors in hCG actions. If receptors are absolutely required, then inhibition of their synthesis should result in a loss of hCG function. We used antisense ODN technology to inhibit receptor synthesis. Immunocytochemical analysis revealed that treatment of endometrial gland epithelial cells with the 21-mer antisense, but not sense, phosphorothioate ODN synthesized from human LH/hCG receptor sequence virtually abolished LH/hCG receptor levels. hCG was not able to increase COX-2 protein, PGE2 secretion, and cAMP production in antisense, but not in sense, ODN-treated cells. Thus, these data clearly demonstrate that hCG cannot work in the absence of a normal complement of its receptors in endometrial gland epithelial cells.

Although the present study cannot vouch for LH/hCG controlling endometrial gland functions in vivo, a recent study demonstrated that hCG can (38). This study has shown that hCG administration in premature ovarian failure patients resulted in morphological changes in endometrial glands that generally reflect functional changes.

Targeted disruption of COX-2, but not COX-1, results in several reproductive defects, one of which is the failure of implantation (21). This finding suggests that COX-2 plays a central role in the implantation process. There must be hormones that up-regulate the expression of COX-2; however, there are no data on what these hormones might be in human endometrial glands. It has long been believed that there is a two-way dialogue between implanting blastocyst and receptive endometrium that is essential for successful implantation. It has been suspected that this dialogue may involve hCG produced by the blastocyst acting on the endometrium (39, 40, 41). This possibility has remained as only a concept, because until recently no one has demonstrated that endometrial glands and stroma contain LH/hCG receptors, and that LH/hCG can increase the expression of COX-2 and, consequently, the much needed eicosanoids that are responsible for all of the other changes that make the endometrium receptive for implantation. This leads to the question of whether there might be other sources of LH/hCG to act on endometrial glands. It is possible that periovulatory blood LH levels may reach concentrations high enough at the endometrial level to begin changes in glands. Endometrial glands produce hCG during secretory phase (42), suggesting that the changes initiated by LH could be continued by hCG derived from glands as well as blastocyst. The present findings also suggest that treatment of woman with hCG to induce ovulation could improve the uterine environment for successful implantation. This benefit could be just as important as the induction of ovulation itself.

It is likely that LH/hCG may also directly and indirectly regulate other endometrial gland functions related to the implantation process. These are, for example, the synthesis and/or secretion of glycodelin, insulin-like growth factor binding protein-1 (43), GnRH (44), and vascular endothelial growth factor (45); the secretion of fluids and electrolytes (46, 47); etc. There is evidence for PGE2 and cAMP regulating some of these functions (46, 47). By increasing PGE2 and cAMP levels, hCG may also regulate these and other endometrial functions.

The present findings may have further implications. For example, coculture of embryos with autologous endometrial epithelial cells has recently been reported to increase the number of blastomeres and decrease the fragmentation rate in preembryos (48). More importantly, coculturing has been found to improve the implantation rate (49), especially in patients who failed to become pregnant in more than three previous cycles in which three or four good quality embryos were transferred (50). The fact that LH/hCG can act on endometrial epithelial cells suggests that hCG treatment may further improve the benefits of coculturing. hCG treatment of women with signs of threatened abortion during early gestation seems to result in less frequent spontaneous abortions, premature deliveries, and intrauterine growth retardation (13), suggesting that hCG improves pregnancy outcome through multiple sites of action, including implantation.

The present findings may also be important for better understanding of endometrial hyperplasias and carcinomas. For example, these tissues overexpress LH/hCG receptors (7, 51), LH and hCG are mitogenic in human endometrial cancer cells (52, 53, 54), and bioactive LH levels are elevated in postmenopausal obese women with endometrial carcinoma compared with those women without this disease (55). If COX-2 is also overexpressed in endometrial hyperplasias and cancers, as in some of the other cancers (56), then further constant stimulation of the enzyme by LH may predispose normal endometrium to become neoplastic. The high PG levels produced from constant COX-2 stimulation can increase cell proliferation, decrease apoptosis, and decrease adhesion to extracellular matrix proteins (56, 57).

In summary, treatment of human uterine endometrial gland epithelial cells with hCG results in a time- and a dose-dependent and hormone-specific increase in COX-2 gene expression. The hCG effect is mediated by the cAMP/type I PKA signaling pathway and requires a normal complement of its receptors in cells. Thus, the effects of hCG and LH may be yet another action of these hormones in human endometrium that is important for implantation of the blastocyst and continuation of pregnancy.

Received February 5, 1999.

Revised April 26, 1999.

Accepted May 20, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Reshef, Lei ZM, Rao Ch V, Pridhan D, Chegini N, Luborsky JL. 1990 The presence of gonadotropin receptors in nonpregnant human uterus, human placenta, fetal membranes and decidua. J Clin Edocrinol Metab. 70:421–430.[Abstract]
  2. Lei ZM, Reshef E, Rao ChV. 1992 The expression of human chorionic gonadotropin/luteinizing hormone receptors in human endometrial and myometrial blood vessels. J Clin Endocrinol Metab. 75:651–659.[Abstract]
  3. Lincoln S, Lei ZM, Rao ChV, Yussman MA. 1992 The expression of human chorionic gonadotropin/human luteinizing hormone receptors in ectopic human endometrial implants. J Clin Endocrinol Metab. 75:1140–1144.[Abstract]
  4. Lei ZM, Rao ChV, Lincoln S, Ackermann DM. 1993 Increased expression of human chorionic gonadotropin/human luteinizing hormone receptors in adenomyosis. J Clin Endocrinol Metab. 76:763–768.[Abstract]
  5. Bhattacharya S, Banerjee J, Sen S, Manna PR. 1993 Human chorionic gonadotropin binding sites in the human endometrium. Acta Endocrinol (Copenh). 129:15–19.[Medline]
  6. Kornyei J, Lei ZM, Rao ChV. 1993 Human myometrial smooth muscle cells are novel targets of direct regulation by human chorionic gonadotropin. Biol Reprod. 49:1149–1157.[Abstract]
  7. Lin J, Lei ZM, Lojun S, Rao ChV, Satyaswaroop PG, Day Jr TG. 1994 Increased expression of luteinizing hormone/human chorionic gonadotropin receptor gene in human endometrial carcinomas. J Clin Endocrinol Metab. 79:1483–1491.[Abstract]
  8. Zuo J, Lei ZM, Rao ChV. 1994 Human myometrial chorionic gonadotropin/luteinizing hormone receptors in preterm and term deliveries. J Clin Endocrinol Metab. 79:907–911.[Abstract]
  9. Toth P, Li X, Rao ChV, Lincoln SR, Sanfilippo JS, Spinnato JA, Yussman MA. 1994 Expression of functional human chorionic gonadotropin/human luteinizing hormone receptor gene in human uterine arteries. J Clin Endocrinol Metab. 79:307–315.[Abstract]
  10. Singh M, Zuo J, Li X, Ambrus G, Lei ZM, Yussman MA, Sanfilippo JS, Rao ChV. 1995 The decreased expression of functional human chorionic gonadotropin/luteinizing hormone receptor gene in human uterine leiomyomas. Biol Reprod. 53:899–904.[Abstract]
  11. Bernardini L, Moretti-Rojas I, Brush M, Rojas FJ, Balmaceda JP. 1995 Status of hCG/LH receptor and G proteins in human endometrium during artificial cycles of hormone replacement therapy. J Soc Gynecol Invest. 2:630–635.[CrossRef][Medline]
  12. Han SW, Lei ZM, Rao ChV. 1997 Homologous down-regulation of luteinizing hormone/chorionic gonadotropin receptors by increasing the degradation of receptor transcripts in human uterine endometrial stromal cells. Biol Reprod. 57:158–164.[Abstract]
  13. Toth P, Gimes G, Paulin F, Rao ChV. 1998 hCG treatment in early gestation: its impact on uterine blood flow and pregnancy outcome [Abstract 0–120]. Fertil Steril. 70:546.
  14. Eta E, Ambrus G, Rao ChV. 1994 Direct regulation of human myometrial contractions by human chorionic gonadotropin. J Clin Endocrinol Metab. 79:1582–1586.[Abstract]
  15. Ambrus G, Rao ChV. 1994 Novel regulation of pregnant human myometrial smooth muscle cell gap junctions by human chorionic gonadotropin. Endocrinology. 135:2772–2779.[Abstract]
  16. Kornyei JL, Li X, Lei ZM, Rao ChV. 1996 Restoration of human chorionic gonadotropin response in human myometrial smooth muscle cells by treatment with follicle stimulating homrone (FSH): evidence for the presence of SH receptors in human myometrium. Eur J Endocrinol. 134:225–231.[Abstract]
  17. Tang B, Gurpide E. 1993 Direct effect of gonadotropins on decidualization of human endometrial stromal cells. J Steroid Biochem Mol Biol. 47:115–121.[CrossRef][Medline]
  18. Han SW, Lei ZM, Rao ChV. 1999 Treatment of human endometrial stromal cells with chorionic gonadotropin induces their morphological and functional differentiation into decidua. Mol Cell Endocrinol. 147:7–16.[CrossRef][Medline]
  19. Han SW, Lei ZM, Rao ChV. 1996 Upregulation of cyclooxygenase-2 gene expression by chorionic gonadotropin during the differentiation of human endometrial stromal cells into decidua. Endocrinology. 137:1791–1797.[Abstract]
  20. Tang B, Guller S, Gurpide E. 1993 Cyclic adenosine 3',5'-monophosphate induced prolactin expression in stromal cells isolated from human proliferative endometrium. Endocrinology. 133:2197–2203.[Abstract]
  21. Lim H, Paria BC, Das SK, Dinchuk JE, Langenbach R, Trzaskos JM, Dey SK. 1997 Multiple female reproductive failures in cyclooxygenase 2-deficient mice. Cell. 91:197–208.[CrossRef][Medline]
  22. Han SW, Lei ZM, Rao ChV. 1996 Upregulation of cyclooxygenase-2 gene expression by chorionic gonadotropin in mucosal cells from human fallopian tubes. Endocrinology. 137:2929–2937.[Abstract]
  23. Rao ChV, Sanfilippo JS. 1997 New understanding in the biochemistry of implantation: potential direct roles of luteinizing hormone and chorionic gonadotropin. Endocrinologist. 7:107–111.
  24. Rao ChV. 1998 Novel concepts in neuroendocrine regulation of reproductive tract functions. In: Bazer FW, ed. The endocrinology of pregnancy. Totowa: Humana Press; 125–144.
  25. Fleming H, Gurpide E. 1982 Growth characteristics of primary cultures of stromal cells from human endometrium. J Steroid Biochem. 16:717–720.[CrossRef][Medline]
  26. Marsh MM, Hampton AL, Riley SC, Findlay JK, Salamonsen LA. 1994 Production and characterization of endothelin released by human endometrial epithelial cells in culture. J Clin Endocrinol Metab. 71:1625–1631.
  27. Sambrook J, Fritsch EF, Maniatis T. 1989 Extraction, purification, and analysis of messenger RNA from eukaryotic cells. In: Nolan C, Ford N, Ferguson M, eds. Molecular cloning: a laboratory manual, ed 2. Cold Spring Harbor: Cold Spring Harbor Laboratory; 7.39–7.57.
  28. Laemmli UK. 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227:680–685.[CrossRef][Medline]
  29. Dunn SD. 1986 Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on western blots by monoclonal antibodies. Anal Biochem. 157:144–153.[CrossRef][Medline]
  30. Keinanen KP, Kellokumpo S, Metsikko MK, Rajaniemi HJ. 1987 Purification and partial characterization of rat ovarian lutropin receptor. J Biol Chem. 262:7920–7926.[Abstract/Free Full Text]
  31. Rao ChV, Griffin LP, Carman Jr FR. 1977 Gonadotropin receptors in human corpora lutea of the menstrual cycle and pregnancy. Am J Obstet Gynecol. 128:146–153.[Medline]
  32. Steel RGD, Torrie JH. 1960 Principles and procedures of statistics, with special reference to the biological sciences. New York: McGraw-Hill.
  33. Cho-Chung YS. 1990 Role of cyclic AMP receptor proteins in growth, differentiation, and suppression of malignancy: New approaches to therapy. Cancer Res. 50:7093–7100.[Abstract/Free Full Text]
  34. Rao ChV. 1996 The beginning of a new era in reproductive biology and medicine: expression of low levels of functional luteinizing hormone/human chorionic gonadotropin receptors in nongonadal tissues. J Physiol Pharmacol. 47:41–53.
  35. Rao ChV, Chegini N, Lei ZM. 1989 Immunocytochemical localization of 5- and 12-lipoxygenases and cyclooxygenase in nonpregnant human uteri. In: Zor U, Naor Z, Danon A, eds. Leukotrienes and prostanoids in health and disease. New trends in lipid mediators research. Basel: Karger; 283–286.
  36. Kennedy TG. 1983 Embryonic signals and the initiation of blastocyst implantation. Aust J Biol Sci. 36:531–543.[Medline]
  37. Samuelsson B, Goldyne M, Granström E, Hamberg M, Hämmarstrom S, Malmsten C. 1978 Prostaglandins and thromboxanes. Annu Rev Biochem. 47:997–1029.[CrossRef][Medline]
  38. Fanchin R, Peltier E, Righini C, Olivennes F, Frydman R, de Ziegler D. 1998 Direct effects of hCG on the human endometrium [Abstract F288]. J Soc Gynecol Invest. 5:109A.
  39. Heap RB, Flint APF, Gadsby JE. 1979 Role of embryonic signals in the establishment of pregnancy. Br Med Bull. 35:129–134.[Free Full Text]
  40. Joshi NJ, Nandedkar TD. 1984 Effects of intrauterine instillation of antiserum to hCG during early pregnancy in mice. Acta Endocrinol (Copenh). 107:268–274.[Medline]
  41. Khan-Dawood FS, Dawood MY. 1984 Chorionic gonadotropin receptors and immunoreactive chorionic gonadotropin in implantation of the rabbit blastocyst. Am J Obstet Gynecol. 148:359–365.[Medline]
  42. Wolkersdörfer GW, Bornstein SR, Hilbers U, Zimmermann G, Biesold C, Lehmann M, Alexander H. 1998 The presence of chorionic gonadotropin ß subunit in normal cyclic human endometrium. Mol Human Reprod. 4:179–184.[Abstract/Free Full Text]
  43. Fazleabas AT, Donnelly KM, Hild-Petito S, Hausermann HM, Verhage HG. 1997 Secretory proteins of the baboon (Papio anubis) endometrium: regulation during the menstrual cycle and early pregnancy. Hum Reprod Update. 3:553–559.[Abstract/Free Full Text]
  44. Raga F, Casan EM, Kruessel JS, Wen Y, Huang H-Y, Nezhat C, Polan ML. 1998 Quantitative gonadotropin-releasing hormone gene expression and immunohistochemical localization in human endometrium throughout the menstrual cycle. Biol Reprod. 59:661–669.[Abstract/Free Full Text]
  45. Hornung D, Lebovic DI, Shifren JL, Vigne JL, Taylor RN. 1998 Vectorial secretion of vascular endothelial growth factor by polarized human endometrial epithelial cells. Fertil Steril. 69:909–915.[CrossRef][Medline]
  46. Vetter AE, Deachapunya C, O’Grady SM. 1997 Na absorption across endometrial epithelial cells is stimulated by cAMP-dependent activation of an inwardly rectifying K channel. J Membr Biol. 160:119–126.[CrossRef][Medline]
  47. Deachapunya C, O’Grady SM. 1998 Regulation of chloride secretion across porcine endometrial epithelial cells by prostaglandin E2. J Physiol. 508:31–47.[Abstract/Free Full Text]
  48. Barmat LI, Liu H-C, Spandorfer SD, Xu K, Veeck L, Damario MA, Rosenwaks Z. 1998 Human preembryo development on autologous endometrial coculture vs. conventional medium. Fertil Steril. 70:1109–1113.[CrossRef][Medline]
  49. Jayot S, Parneix I, Verdaguer S, Discamps G, Audebert A, Emperaire JC. 1995 Coculture of embryos on homologous endometrial cells in patients with repeated failures of implantation. Fertil Steril. 63:109–114.[Medline]
  50. Simon C, Mercader A, Garcia VJ, Valbuena D, Herrer R, Moreno C, Pellicer A. 1998 Coculture of embryos with autologous human endometrial epithelial cells and transfer at blastocyst stage increases implantation rates in patients with repeated failures of implantation[Abstract 0–057]. Fertil Steril. 70:522.
  51. Konishi I, Koshiyama M, Mandai M, et al. 1997 Increased expression of LH/hCG receptors in endometrial hyperplasia and carcinoma in anovulatory women. Gynecol Oncol. 65:273–280.[CrossRef][Medline]
  52. Bax CMS, Chatzaki E, Davies S, Gallagher CJ. 1996 Elucidating the role of gonadotropins in endometrial cancer cell growth. Biochem Soc Trans. 24:443S.
  53. Nagamani M, Cao HA. 1997 Specific binding and proliferative effects of luteinizing hormone in human endometrial cancer cell lines [Abstract 211]. J Soc Gynecol. Invest 4:132A.
  54. Han SW, Zhou XL, Lei ZM, Rao ChV. Role of luteinizing hormone in human endometrial carcinoma. 81st Annual Meeting of The Endocrine Society, San Diego, CA, 1999, in press.
  55. Nagamani M, Doherty MG, Smith ER, Yallampalli C. 1992 Increased bioactive luteinizing hormone levels in postmenopausal women with endometrial cancer. Am J Obstet Gynecol. 167:1825–1830.[Medline]
  56. Subbaramaiah K, Zakim D, Weksler BB, Dannenberg AJ. 1997 Inhibition of cyclooxygenase: a novel appraoch to cancer prevention, Proc Soc Exp Biol Med. 216:201–210.[Abstract]
  57. Tsujii M, DuBois RN. 1995 Alterations in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase 2. Cell. 83:493–501.[CrossRef][Medline]



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