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. 2008 Oct 23;150(3):1512–1520. doi: 10.1210/en.2008-1081

Facilitative Glucose Transporter Type 1 Is Differentially Regulated by Progesterone and Estrogen in Murine and Human Endometrial Stromal Cells

Antonina Frolova 1, Lauren Flessner 1, Maggie Chi 1, Sung Tae Kim 1, Nastaran Foyouzi-Yousefi 1, Kelle H Moley 1
PMCID: PMC2654750  PMID: 18948400

Abstract

Embryo implantation is a highly synchronized event between an activated blastocyst and a receptive endometrium. The success of this process relies on the dynamic interplay of estrogen (E2) and progesterone (P4), however, the details of this interaction are not entirely clear. Recent data implicate E2 and P4 in the regulation of glucose utilization by affecting facilitative glucose transporter (GLUT) expression. In this study we examine GLUT1 expression in murine and human endometrial stromal cells (ESCs) using a primary culture system. We show that expression of GLUT1 is increased during ESC decidualization in vitro. P4 up-regulates, whereas E2 down-regulates, GLUT1 expression. In addition, P4 increases and E2 decreases glucose uptake in ESCs, suggesting that GLUT1 may be a major player in glucose utilization in these cells. Moreover, GLUT1 expression is increased in human ESCs when decidualized in vitro with P4 and dibutyryl cAMP, suggesting a similar role for P4 in human endometrium. In conclusion, an imbalance between P4 and E2 seen in patients with polycystic ovary syndrome, luteal phase defect, and recurrent pregnancy loss may have a critical impact on glucose utilization in the endometrial stroma, and, thus, may be responsible for endometrial dysfunction and failure of embryo implantation in these patient populations.

GLUT1 expression increases during decidualization of endometrial stromal cells in vitro. Progesterone upregulates and estrogen downregulates GLUT1 expression and glucose uptake in these cells.

Recurrent pregnancy loss (RPL) affects about 1% of couples attempting to conceive, and over 50% of these cases are of unknown etiology (1). For many years RPL and infertility were attributed primarily to a luteal phase defect (LPD), caused by a decreased level of progesterone (P4) secreted by the corpus luteum. Historically, it was diagnosed by an endometrial biopsy, and, although there is controversy and lack of strong evidence supporting LPD as a cause of RPL, P4 supplementation remains a standard treatment for the disorder, and a significant percentage of women demonstrate a reduction in miscarriage rates (2,3).

However, an equal number of studies show no beneficial effect of supplemental P4, most likely because many factors are involved in mediating embryo implantation. The process is a sophisticated and highly coordinated event between an activated preimplantation embryo and a receptive endometrium, and as a result, in most mammals, implantation can only occur during a restricted time termed the “window of implantation” (4). Endometrial receptivity for implantation occurs independently of the embryo. Therefore, even if development of the embryo proceeds normally, implantation failure and pregnancy loss can occur due to endometrial dysfunction. This window of implantation in the uterus is determined primarily by the dynamic interplay of the sex steroids, estrogen (E2) and P4 (5). However, the precise mechanisms of regulation and the degrees of independent contribution by each hormone are still unknown (6). In general, P4 is required by all mammalian species for implantation and is the dominant hormone during the secretory phase in the uterus, when endometrial morphology transforms to prepare a receptive environment for an embryo (7). The role of E2 in this process is more complex (5). Although it is known that implantation in humans and nonhuman primates depends on the actions of P4 in an E2-primed uterus (8), the precise role of E2 in the development of a receptive endometrium in these species is still unclear.

The endometrium is composed of several major cell types that include epithelial, endothelial, stromal, and nonresident immune cells. Each of these cell types plays a different role in implantation. Although the epithelial cells are important for initial blastocyst adhesion, the endometrial stromal cells (ESCs) play the most critical role during embryo invasion into the uterine wall (9). At implantation, mammalian ESCs undergo a drastic functional and morphological change referred to as decidualization. These decidual cells are believed to provide the implanting embryo with a number of factors required for implantation and development (4). Moreover, the extent of decidualization correlates with the depth of trophoblast invasion and placentation in different mammalian species (10). Thus, we suspect that impaired ESC decidualization will likely result in poor embryo implantation and subsequent pregnancy loss.

Although P4 is the main player in uterine stroma decidualization in nonhuman primates, high E2 activity can also cause problems with this process (8). Several studies have suggested a direct link between high E2 levels or E2 receptor (ER) activity and increased incidence of miscarriages. Women with endometriosis, which often leads to subfertility, demonstrate increased expression of ERα in the endometrium and often lack integrinβ3, a biomarker of uterine receptivity (11). Ovarian hyperstimulation syndrome in in vitro fertilization (IVF) practices provides further support that abnormal E2 levels may play a role in miscarriages. Patients exhibit increased numbers of growing follicles and retrievable oocytes due to extremely high E2 levels, but they also experience an increased abortion rate and decreased implantation rates (12). Similarly, ERα overexpressing mice demonstrate a decreased litter size (13). These studies imply that the correct balance between the two sex steroids may be just as important as the level of either hormone alone in determining uterine receptivity at implantation.

Recent studies have begun to implicate steroid hormones, E2 and P4, in the regulation of glucose metabolism by regulating glucose transporter (GLUT) expression. Currently, there are 13 described members of the facilitative GLUT family, GLUTs 1-12 and the H+-coupled myo-inositol-transporter (HMIT). These transporters exhibit a high degree of sequence homology but differ in tissue and subcellular distribution, kinetics, and substrate specificity (14,15,16). The GLUTs are responsible for glucose uptake by cells and, thus, play a critical role in normal glucose utilization. GLUT1 was up-regulated by either E2 or P4 in Ishikawa endometrial cancer cells, whereas combined E2 and P4 treatment reversed this effect (17). Interestingly, in a different breast cancer cell line, ZR-75-1, GLUT1 was up-regulated by P4 and a combined treatment of P4 and E2, but not by E2 alone (18). In this cell line, GLUT4 was also up-regulated by all three hormonal treatments. In addition, GLUT1 and GLUT3 are known to be expressed in human endometrium, and GLUT1 expression increased upon decidualization of the uterine stromal cells in vitro (19).

In this work we examined GLUT1 protein expression in ESCs using primary culture systems of murine and human ESCs (hESCs). In both culture systems, we examined GLUT1 protein regulation during the process of ESC decidualization in vitro. In the murine ESCs, we further investigated the individual effects of P4 and E2 on GLUT1 expression and 2-deoxyglucose (DG) uptake.

Materials and Methods

Animal care and use

All mouse studies were approved by the Animal Studies Committee at Washington University School of Medicine and conform to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Female C57BL/6NCr (National Cancer Institute, Bethesda, MD) mice 7–8 wk of age were used in these experiments. For studies involving pregnant mice, the day on which a vaginal plug was detected is referred to as the first day postcoitus (dpc).

Isolation of murine ESCs and culture conditions

For in vitro decidualization of ESCs, female mice were killed at 4 dpc. Pregnancy was confirmed by flushing dissected uterine horns and ostia with Hanks’ balanced salt solution, and observing for the presence of blastocysts. Uteri confirmed for pregnancy were cut into 1-mm3 pieces and incubated in DMEM:F12 without phenol red supplemented with 2 g/liter Collagenase Type I (Life Technologies, Inc., Gaithersburg, MD) for 60–90 min at 37 C. Tissues were vortexed every 15 min during incubation. After incubation the solution was passed through a 40-μm sieve (BD Falcon, BD Biosciences, San Jose, CA) and centrifuged at 1100 rpm for 5 min. The ESCs were then resuspended in DMEM:F12 without phenol red supplemented with 2% heat-inactivated charcoal dextran-stripped calf serum (HyClone, Logan, UT) and 50 μg/ml penicillin/streptomycin (Cambrex Bio Science, Walkersville, MD). ESCs were plated in six-well cell culture plates at 7.5 × 105 cells per well, and media were supplemented with β-estradiol water-soluble (E2) at a final estradiol concentration of 10 nm and P4-water soluble at a final P4 concentration of 1 μm for 72 h (Sigma-Aldrich Corp., St. Louis, MO). Control samples received no hormone supplementation.

Isolation of hESCs and culture conditions

Endometrial tissue was obtained from human uteri after hysterectomy conducted for benign disease or from endometrial biopsies. Informed consent was obtained from each patient before surgery, and protocols were approved by the Human Research Protection Office of Washington University (no. 07-0949). Tissues were placed in Hanks’ balanced salt solution and transported to the laboratory. hESCs were isolated and cultured as previously described (20). In vitro decidualization was performed as previously described (21) with several changes. Cells were seeded in six-well plates at 2 × 10−5 cells per well in DMEM:F12 without phenol red supplemented with 2% heat-inactivated charcoal dextran-stripped calf serum and antibiotics. Cells were treated with 1 μm medroxyprogesterone-17-acetate (MPA) (Sigma-Aldrich) and 0.5 mm N6, 2′-O-dibutyryl cAMP (db-cAMP) (Sigma-Aldrich). Control samples received 0.1% ethanol vehicle control.

Western blot analysis

Cells were lysed in 150 μl lysis buffer, 50 mm Tris HCl (pH 7.5), 150 mm NaCl, 5 mm EDTA, 0.5% Nonidet P-40, and complete Mini protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN), per well. Protein concentration was quantified using the BCA Protein Assay Kit (Pierce, Rockford, IL). Whole cell lysates were then separated using SDS-PAGE and transferred to nitrocellulose. Nonspecific antibody binding was blocked in 5% nonfat dry milk powder in 1× Tris-buffered saline for 1 h. Blots were incubated in 1% nonfat dry milk powder in Tris-buffered saline with 0.05% Tween 20 overnight at 4 C with the following antibodies: rabbit anti-GLUT1 (1:2000) (kindly provided by Dr. Michael Mueckler, Washington University) (22); rabbit anti-GLUT4 (1:1000) (kindly provided by Dr. Michael Mueckler, Washington University) (23); rabbit anti-GLUT8 (1:1000) (previously generated in our laboratory) (24); rabbit anti-GLUT9b (1:1000) (previously generated in our laboratory) (25); rabbit anti-GLUT12 (1:1000) (generated in our laboratory and characterized as monospecific, unpublished data); and mouse antiactin (1:1000) (CHEMICON International, Inc., Temecula, CA). For experiments represented in Figs. 1, 2, and 3, blots were incubated with horseradish peroxidase-conjugated goat-antirabbit or goat-antimouse secondary antibody (1:10,000) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h at room temperature. GLUT1, GLUT9b, and β-actin were detected using ECL Western blotting detection reagents (Amersham Biosciences Inc., Piscataway, NJ). GLUT8 and GLUT12 were detected using SuperSignal Dura chemiluminescence substrate (Pierce). For experiments represented in Figs. 4, 5, and 6, blots were incubated with goat-antimouse IRDye 680 or goat-antirabbit IRDye 800 (1:10,000) (LI-COR Biosciences, Lincoln, NE) for 1 h at room temperature. Membranes were scanned and quantitated using the Odyssey fluorescent imager (LI-COR Biosciences). Western blots were quantified, and the values were normalized to β-actin for loading variation.

Figure 1.

Figure 1

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Expression of facilitative GLUTs in murine ESCs decidualized in vitro. A, ESCs were isolated from mice on d 4 pregnancy and cultured for 72 h in the presence of 1 μm P4 and 10 nm E2 to induce decidualization. Control cells were cultured without hormones. GLUT1 protein expression was quantitated by Western blot and normalized to the internal control, β-actin, for loading variability. Fold change compared with the “Control” value is represented in the graph. Values are the mean ± sem of three independent experiments. *, P < 0.001. B, Representative Western blot demonstrates GLUT1 expression and the unchanged loading control, β-actin. C, ESCs cultured in vitro with hormones (“D”) or without (“C”). GLUT1 protein expression was analyzed after 24, 48, and 72 h culture. A representative Western blot is shown. GLUT1 begins to increase after the initial 24 h E2 and P4 treatment in decidualized ESCs from pregnant mice, and the increase is sustained at 48 and 72 h. D, Protein levels of GLUT4, GLUT8, GLUT9b, and GLUT12 were analyzed by Western blot in decidualized ESCs compared with control. GLUT proteins have a high degree of N-linked glycosylation and, thus, appear as multiple bands or heterogeneous bands when subjected to SDS-PAGE analysis (35). E, qPCR was used to monitor levels of Prp mRNA, a decidualization marker, in ESCs after 72 h culture.

Figure 2.

Figure 2

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GLUT1 protein expression is differentially regulated by P4 and E2 in murine ESCs. ESCs were isolated from uteri of nonpregnant mice and cultured in phenol red-free media for 4 d before hormone treatment. A, Cells were then plated at 5 × 105 cells per well in six-well cell culture plates and cultured for 72 h in the presence of 10 nm E2, 1 μm P4, or both 10 nm E2/1 μm P4. GLUT1 protein expression was analyzed by Western blot and normalized to the internal control, β-actin, for loading variability. Fold change compared with the “Control” value is represented in the graph. C, ESCs were also treated with 1 μm RU486, a PR antagonist. B and D, Representative Western blots show relative expression of GLUT1 and the loading control β-actin. Values are the mean ± sem of three independent experiments. *, P < 0.001 compared with control by ANOVA with Fisher’s test. E, ESCs were isolated from uteri of nonpregnant mice, maintained in phenol red-free media for 4 d, and then treated with 1 μm P4 for the durations indicated. mRNA expression was analyzed by qPCR, and samples were normalized to a housekeeping gene, Gapdh, for a loading control. Values are a mean ± sem of four independent experiments. *, P < 0.005 compared with 0 h by ANOVA with Fisher’s test. F, GLUT1 protein expression was analyzed by Western blot. Values are a mean ± sem of three independent experiments. *, P < 0.005; **, P < 0.05 compared with the 0 h sample by ANOVA with Fisher’s test. G, A representative Western blot showing GLUT1 expression.

Figure 3.

Figure 3

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GLUT1 localizes to the plasma membrane. A, Cell surface GLUTs were labeled by incubating ESCs with Bio-ATB-BMPA and exposing the mixture to UV light. Cells were then solubilized for immunoprecipitation with streptavidin-agarose beads. Cell surface GLUT1 expression was analyzed by Western blot. To determine whether differences in cell surface expression levels of GLUTs among the treatment groups were attributable to differences in GLUT expression levels or in GLUT localization, the total expression levels of GLUTs in each sample was also analyzed by Western blotting and normalized to β-actin for loading variability. B, A representative Western blot of GLUT1 cell surface expression is shown. C, A representative Western blot of total GLUT1 expression is shown. D, ESCs isolated from the uteri of pregnant mice were decidualized in vitro. Cell surface GLUTs were labeled by incubating ESCs with Bio-ATB-BMPA and then solubilized for immunoprecipitation with streptavidin-agarose beads. Cell surface GLUT1 expression and total GLUT1 expression were analyzed by Western blot. E, A representative Western blot of cell surface GLUT1 expression in ESCs isolated from mice at 4 dpc is shown. F, A representative Western blot of total GLUT1 in ESCs isolated from mice at 4 dpc is shown. Values are the mean ± sem of six samples analyzed in each treatment group. *, P < 0.001 compared with control by ANOVA with Fisher’s test. G, GLUT1 localization in vivo in the uterine decidual cells at 7 dpc. Uteri were collected from five mice, and a representative section is shown.

Figure 4.

Figure 4

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P4 increases glucose uptake in ESCs. DG uptake in ESCs cultured in the presence of P4, E2, or P4+E2 for 72 h. Uptake was measured after 60 min incubation with 0.1 mm DG, and each sample was normalized to total protein. Values are mean ± sem of six samples analyzed in each treatment group. *, P < 0.001 compared with vehicle control by ANOVA with Fisher’s test.

Figure 5.

Figure 5

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GLUT1 is expressed in vivo in ESCs at pregnancy. A, ESCs were isolated from mice on the indicated day of pregnancy, and protein expression of GLUT1 was analyzed by Western blot. Values are a mean of five mice. *, P < 0.05 by ANOVA with Fisher’s test. B, Western blot showing GLUT1 expression and the loading control, β-actin. C, GLUT1 expression at 5 dpc by immunohistochemistry in the implantation site (IS) and interimplantation site (Inter-IS). Five mice were used, and a representative section is shown. The protein can be seen in the decidualizing stroma (s) close to the embryo (em), as well as the glandular and luminal epithelium (ge and le, respectively). D, By 7 dpc, GLUT1 expression can be seen primarily in the growing decidualizing stroma in the implantation site surrounding the embryo (em) and no longer in the epithelial tissues. Five mice were used, and a representative section is shown.

Figure 6.

Figure 6

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GLUT1 expression increases in hESCs during decidualization in vitro. A, GLUT1 protein expression increases significantly in hESCs after 3, 6, and 9 d 1 μm MPA and 0.5 mm db-cAMP treatment in vitro. Fold changes are calculated compared with the 3 d untreated control sample. Values are a mean of three independent experiments ± sem. *, P < 0.05; **, P < 0.001 by ANOVA with Fisher’s test. B, Representative Western blot of GLUT1 and the loading control β-actin showing protein lysates from control (“C”) and decidualized (“D”) hESCs collected after the indicated length of treatment time. C, qPCR was used to monitor levels of Igfbp1 mRNA, a decidualization marker in hESCs after 3, 6, and 9 d culture. Samples are normalized to the untreated-control sample on the appropriate days. D, qPCR was used to monitor levels of Prl mRNA, a decidualization marker in hESCs after 3, 6, and 9 d culture. Samples are normalized to the untreated-control sample on the appropriate days.

Quantitative RT-PCR (qPCR)

Total RNA was isolated using TRIzol reagent (Invitrogen Corp., Carlsbad, CA) and deoxyribonuclease-treated with the DNA-free kit (Ambion, Inc., Austin, TX). One microgram of RNA was reverse transcribed using SuperScript III and Oligo(deoxythymidine) primers (Invitrogen) in a total of 20 μl. qPCR was preformed using SYBR green fluorescence and an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). Each reaction was run in triplicate and consisted of 50 ng cDNA, 1 × Power SYBR Green PCR System (Applied Biosystems) and 300 nm of the primers listed in supplemental Table S1, which is published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org. The fold change in gene expression was calculated using the ΔΔCt method with the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase, as the internal control in murine cell studies and ribosomal protein, 36B4, the internal control in human cell studies.

Cell surface photolabeling of GLUTs

ESCs derived from pregnant or nonpregnant mice were cultured and treated with steroid hormones as described previously. ESCs were then analyzed for cell surface GLUTs as previously described, using the biotin membrane impermeant reagent N-[2-[2-[2- [N-biotinylcaproylamino]-ethoxy]-ethoxyl]-4-[2- (trifluoromethyl)-3H-diazirin-3yl] benzoyl]-1, 3-bis(mannopyranosyl-4-yloxy)-2-propylamine (Bio-ATB- BMPA) (26). Plasma membrane extracts were isolated by centrifugation, and protein concentration was quantified using the BCA Protein Assay Kit. An equal amount of protein for each sample was added to streptavidin-agarose beads for immunoprecipitation. After an overnight incubation with streptavidin beads, bound GLUTs were eluted and analyzed by Western blot.

Immunofluorescence

Tissues were fixed in 4% paraformaldehyde for 30 min, flash frozen in liquid nitrogen, and sectioned at 7 μm. Tissues were postfixed in methanol, and washed in PBS for 5 min and blocked for 60 min in 1% BSA/0.3% Triton X-100/5% normal goat serum in PBS. The sections were incubated with rabbit anti-GLUT1 (referenced in Western blot analysis) at 20 μg/ml for 60 min, washed three times for 5 min with PBS, and incubated with Alexa Fluor 488 goat antirabbit (1:250; Molecular Probes, Inc., Eugene OR) for 30 min. Sections were counterstained with TO-PRO-3 iodide dye (Molecular Probes). Fluorescence was observed under a confocal microscope (Nikon Eclipse E800; Nikon Corp., Tokyo, Japan). Negative control slides were immunolabeled with an equal concentration of rabbit IgG.

DG uptake assay

ESCs were isolated from nonpregnant mice as described previously. The DG uptake assays have been previously described in detail by our laboratory using the preimplantation embryo as the model (27). We have adapted this assay for ESCs in the following manner. Primary ESC cultures were plated at 5 × 105 cells in six-well plates. After the treatments described previously, cells were incubated for 60 min in a simple salt solution (125 mm NaCl, 4.7 mm KCl, 2.5 mm CaCl2, 2.4 mm MgSO4, 25 mm NaHCO3, and 1.2 mm K2HPO4) without glucose, supplemented with 1% BSA, at 37 C and 5% CO2. Next, cells were incubated in 100 μm DG for 60 min at 37 C and 5% CO2. The glucose uptake was stopped by incubating the cells in 50 μm cytochalasin B for 1 min. After dissociation from the plate using Cell Dissociation Solution (Sigma-Aldrich), cells were homogenized in 100 μl 0.05 n NaOH. Twenty-five microliters of 0.18 n HCl were added to a 50-μl aliquot of 0.05 n NaOH to maintain a 27-mm excess, and the homogenate was heated to 80 C for 20 min, and then 25 μl 0.16 m Tris base was added to maintain a pH of 8.1. The samples were stored at −80 C until the microanalytical assays were performed to determine the DG levels. Reactions are normalized to total protein, and DG concentrations are expressed as mmoles/kg · 60 min protein.

Immunohistochemistry

Frozen sections (10 μm) were fixed in 3% paraformaldehyde for 15 min and washed two times. After blocking the endogenous peroxidase activity with 3% H2O2 in methanol for 10 min and blocking background with 10% nonimmune goat serum for 1 h, the sections were incubated with the primary antibody (10 μg/ml) at 4 C overnight, washed three times for 5 min with PBS, and incubated with biotinylated secondary antibody (Zymed Laboratories, Inc., San Francisco, CA) for 30 min. Sections were then incubated with the enzyme conjugate (streptavidin peroxidase; Zymed Laboratories) for 30 min. The coloring reaction was done using 3,3′-diaminobenzidine, and sections were counterstained with hematoxylin (Zymed Laboratories).

Statistical analyses

Differences between control values and experimental values were compared by the Student’s t test or ANOVA with Fisher’s post hoc test when comparisons were made between more than one experimental group (StatView 4.5; SAS Institute Inc., Cary, NC).

Results

GLUT1 expression is up-regulated in murine ESCs during decidualization in vitro

Preliminary data in our laboratory and work from other groups (19) strongly suggested that expression of facilitative GLUTs may be regulated in the endometrial stroma at embryo implantation. Therefore, to understand further this phenomenon and its biological significance, we used a previously described murine primary culture system (28) to study whether expression of any of the members of the GLUT family increased during decidualization of ESCs. ESCs isolated from pregnant mice were cultured in vitro in the presence of E2 and P4 for 72 h. Decidualization was confirmed by monitoring prolactin-related protein (Prp) mRNA expression by qPCR (Fig. 1E). This induction of Prp expression was previously demonstrated in decidualized ESCs at 72 h treatment (29). Next, we examined the levels of several GLUTs by Western immunoblot and found that GLUT1 protein expression was 2-fold higher in the decidualized ESCs compared with the control ESCs (Fig. 1, A and B). The increase in GLUT1 protein was first detected as early as 24 h hormone treatment (Fig. 1C). In addition, we evaluated the levels of GLUT4, GLUT8, GLUT9b, and GLUT12. All of these transporters were present in the ESCs but did not exhibit up-regulation upon decidualization. Interestingly, the GLUT12 concentration was found to decrease during decidualization (Fig. 1D).

P4 and E2 differentially regulate GLUT1 protein expression in ESCs in vitro

We hypothesized that GLUT1 may be directly regulated by either E2 or P4 in the uterine stroma in vitro. To eliminate the possibility that the changes we observed in GLUT1 expression were the result of the decidualization process and not a direct affect of the hormones themselves, we used primary ESC cultures isolated from nonpregnant mice in the following experiments and dissected the hormonal effects on GLUT1 expression. As indicated in Fig. 2A, GLUT1 protein levels increased 2-fold with P4 treatment, and this effect was reversed upon addition of E2. RU486, a well-characterized progesterone receptor (PR) antagonist, also reversed the effect of P4 on GLUT1 protein expression (Fig. 2C), suggesting that the regulation is likely achieved at the transcriptional level and is mediated by the PR. We next examined the kinetics of P4 effects on GLUT1 expression. Glut1 mRNA levels started to increase at 8 h P4 treatment and peaked at 24 h (Fig. 2E). The protein levels showed a delayed response, with the first increase observed at 24 h (Fig. 2, F and G). The protein levels remain elevated until 72 h. Together, the data demonstrate that P4 regulates GLUT1 gene transcription.

Thus, P4 is an activator of GLUT1 expression in the uterine stroma, and E2 antagonizes P4 effects. These data suggest that an imbalance of the sex steroid hormone levels in the uterus at implantation may have a drastic effect on glucose utilization in the uterine stroma and, ultimately, on the implantation process.

P4 treatment leads to increased GLUT1 expression at the plasma membrane

To assess whether the P4-induced up-regulation of GLUT1 expression results in its higher accumulation in the appropriate cellular compartments, primarily the plasma membrane, we assessed its cellular localization in primary cultures of ESCs by exofacial photolabeling with Bio-ATB-BMPA, immunoprecipitating with streptavidin-agarose beads and analyzing GLUT1 by Western blot (26,30). After 72 h P4 treatment in vitro, GLUT1 protein expressed on the surface of ESCs from nonpregnant mice increased approximately 2-fold, and this effect was reversed with E2 treatment (Fig. 3, A and B). This increase in surface GLUT1 protein expression correlated with the increase in total GLUT1 protein, indicating that the newly expressed GLUTs were transported to the cell surface. These results were also confirmed with cell fractionation experiments (supplemental Fig. S1). The levels of GLUT1 at the plasma membrane were also higher in the decidualized ESCs compared with control, suggesting an increase in functional GLUT1 during this stromal differentiation in vitro (Fig. 3, D and E). We also examined localization of GLUT1 by immunofluorescence in vivo in decidualized ESCs at d 7 pregnancy. GLUT1 was localized to the plasma membrane, confirming our in vitro results (Fig. 3G). In contrast to GLUT1, the amount of GLUT4, GLUT8, and GLUT9b on the plasma membrane did not increase (data not shown), which eliminated the possibility that P4 or E2 induced nonspecific trafficking of GLUTs to the plasma membrane.

DG uptake correlates with GLUT1 protein levels

To establish a correlation between GLUT1 protein levels and glucose utilization in the endometrial stroma, we examined the efficiency of glucose uptake by using our ESC primary culture system. We used microfluorometric enzyme assays to evaluate the uptake of DG. ESCs were isolated from nonpregnant mice and treated with vehicle, E2, P4, or E2 plus P4. After 72 h treatment, DG uptake was measured. Compared with the vehicle control, the DG uptake significantly increased during P4 treatment (Fig. 4). This higher level of DG uptake correlated with the aforementioned increase in GLUT1 expression (Fig. 2A).

Interestingly, the DG uptake was brought below basal levels when the stromal cells were incubated concurrently with E2 and P4 or with E2 alone (Fig. 4). This negative effect of E2 treatment on glucose uptake in the ESCs suggests that this hormone appears to play a significant role in regulation of glucose metabolism, and that an imbalance in the P4 and E2 levels in the uterus may have functional effects on glucose utilization in the uterine stroma.

GLUT1 expression increases during pregnancy in murine ESCs in vivo

To assess whether the effects we observed in our murine primary culture system correctly reflect the physiology of endometrial stroma during pregnancy, we evaluated the levels of GLUT1 in murine ESCs in vivo. ESCs were isolated from the uteri of mice during pregnancy at 4–7 dpc. It is known that the murine embryo implants on 4.5 dpc and decidualization of the endometrial stroma begins around the same time. We analyzed levels of GLUT1 protein and observed that expression was low on 4 dpc, before embryo implantation, but significantly increased at 7 dpc (Fig. 5, A and B). Although not statistically significant, GLUT1 protein levels began to increase as early as 5 dpc. These results support our in vitro decidualization. We also assessed the localization of GLUT1 in murine implantation sites by immunohistochemistry at d 5 and 7 pregnancy. At d 5, GLUT1 was present in the glandular and luminal epithelium, and in the decidualizing stroma directly around the embryo (Fig. 5C). By d 7, GLUT1 was present mostly in the decidualized stroma of the implantation site (Fig. 5D). The expression of GLUT1 was significantly lower in the interimplantation site stroma, suggesting a pregnancy associated role for GLUT1 in the uterus.

GLUT1 expression increases in hESCs during in vitro decidualization

To assess the possible significance for our novel findings with respect to human physiology, we applied a primary culture system of hESCs. The hESCs were isolated, cultured in phenol red-free media, and then treated with MPA and db-cAMP to induce decidualization. The expression of decidualization markers Prl and Igfbp1 progressively increased on d 3, 6, and 9 treatment, confirming decidualization (Fig. 6, C and D). By 3 d treatment, in good correlation with the aforementioned described data, GLUT1 protein levels began to increase compared with control hESCs, and was significantly higher on d 6 and 9 treatment (Fig. 6, A and B). The mRNA levels of Glut1 also increased by d 3 in the decidualized ESC samples (data not shown). This was a strong indication that the regulation detected in the mouse model of stromal decidualization might also apply to the human stroma. Because the decidualization of hESCs in vitro is induced by treatment with only MPA and db-cAMP, in the absence of any E2, the resulting increase in GLUT1 protein is most likely a P4 effect.

Discussion

The process of embryo implantation in the uterus requires proper differentiation of the endometrium in a time-appropriate manner. The ESCs play a key role in embryo invasion into the uterus, and undergo a morphological and functional change termed decidualization to form decidual cells. The decidual cells provide the environment required for blastocyst invasion and further development. This process is mediated mainly by P4 and, to a certain degree, E2. Because recent studies have shown that these steroid hormones play a role in glucose utilization by altering expression of several facilitative GLUTs in human breast cancer cell lines and murine muscle and adipose tissue (17,18,31,32), we used a primary culture of murine ESCs to examine the possible effects of P4 and E2 on glucose utilization in these cells.

We selected several candidate GLUTs and examined their expression levels before and after ESC decidualization in vitro. Our findings show that GLUT1 expression is significantly increased during ESC decidualization in the pregnant mouse uterus, whereas GLUT4, GLUT8, GLUT9b, and GLUT12 were not increased. In contrast, GLUT12 protein levels decreased drastically upon decidualization in vitro. We suspect that this protein may play a role before decidualization, and ongoing studies are aimed at examining its regulation and role in ESCs. Because GLUT1 was the only transporter that showed marked increase in expression at decidualization, we focused further studies on its regulation. We found that GLUT1 up-regulation is mediated by P4 acting through PR and is reversed by the antagonist, RU486.

The possibility existed that GLUT1 was up-regulated but nonfunctional, needing an additional signal for activation. To determine the functional significance of this increase in GLUT1 protein expression, we determined the cellular localization of the protein as well as DG uptake in ESCs. Our localization studies confirmed that the newly expressed GLUT1 protein is at the plasma membrane where it most likely serves as a GLUT, both in vitro and in vivo. In addition, we showed that DG uptake increases upon P4 treatment of ESCs, which suggests that GLUT1 is functional and may be the main GLUT in these cells. Further studies need to be conducted to confirm these conclusions.

Interestingly, E2 reverses this effect and brings GLUT1 expression to basal levels in ESCs isolated from nonpregnant mice. The finding that these hormones act in opposition is important because it suggests that a careful balance is needed to achieve proper gene expression profiles required for decidualization. These hormones are both present at implantation, and our study shows that they not only induce interesting individual GLUT expression profiles but also interact to regulate the expression of GLUT1 and, potentially, other genes. A previous study in Ishikawa endometrial cancer cells also showed cross talk between P4 and E2 in regulating GLUT1 expression. The hormones up-regulated expression individually, but the effect was abolished when both steroids were present (17). This study, along with ours, suggests that the mechanism of E2 and P4 cross talk may be important in the regulation of GLUT1 expression at times of increased glucose requirements such as in the decidualizing stroma or cancer cells, which have an increased growth phenotype. Our ongoing and future studies focus on deciphering the mechanism of cross talk between these two hormones, and determining the effects of hormone imbalance on glucose utilization and decidualization in the endometrial stroma.

Besides the differential effects of these hormones on GLUT1 expression, we also saw a differential effect on DG uptake in the murine stromal cells. Although P4 increased DG uptake, E2 alone and E2 plus P4 treatments significantly decreased DG uptake in ESCs. This suggests that other GLUTs are also involved that E2 may induce their down-regulation or inactivation. Indeed, GLUT12 is an example of this possibility because it was down-regulated in decidual ESCs, however, further studies need to be done to assign a functional significance to this transporter. There is also a number of GLUTs that we did not examine in this study, which may be down-regulated by E2. The observation that P4 treatment increases DG uptake in correlation with increased GLUT1 protein supports our hypothesis that GLUT1 may be the main transporter responsible for glucose uptake at implantation in the stromal cells.

Although DG uptake was down-regulated with concurrent E2 plus P4 treatment in our study, we suspect that this effect is only true of the ESCs isolated from nonpregnant mice and is not true for ESCs isolated from the pregnant females, which can be decidualized in vitro. Studies show that P4 is the dominant hormone at implantation, and a down-regulation of ER is a prerequisite for implantation (8). Therefore, we suggest that in the nonpregnant endometrium, GLUT1 expression is kept in check by the balancing effects of E2 and P4. However, during pregnancy, the effects of P4 are increased, due to higher levels of P4 and PR, whereas the effects of ER are decreased. Thus, GLUT1 expression is up-regulated and leads to an increase in glucose uptake upon decidualization. This model also suggests that an imbalance in the sex hormones, such as that seen in women with LPD or polycystic ovary syndrome, may lead to dysregulation in glucose utilization in the uterine stroma and subsequent reproductive failures.

Along with our characterization of GLUT1 regulation and function in the murine endometrial stroma, we noted an increase in this protein in the hESCs in response to decidualization in vitro. We used a primary culture system of hESCs and induced the decidual process in vitro with MPA and cAMP treatment. It has previously been shown that when hESCs were decidualized in vitro with P4 and E2, GLUT1 expression increased starting at 14 d treatment (19). This study also showed that decidualization was interrupted upon treatment with cytochalasin B, a GLUT inhibitor, suggesting that an important role for glucose utilization during hESC decidualization may exist. We modified our decidualization protocol according to data that has shown that P4 alone can induce decidualization through cAMP and protein kinase A signaling (10,33). Treatment of hESCs with P4 increases levels of cAMP and leads to the increased expression of decidualization markers. Moreover, cotreatment with a protein kinase A inhibitor inhibits this phenotype (33). However, P4 and E2 treatment without cAMP leads to only a modest and delayed increase in the decidualization markers. Our results show that when hESCs are exposed to MPA and cAMP, even in the absence of E2, GLUT1 expression begins to increase as early as 3 d treatment and is significantly higher after 6 and 9 d. These data show that GLUT1 expression is dependent only upon MPA and cAMP treatment, without E2 presence, which correlates with our studies in the mouse in which actions of P4 were the mechanism behind increased GLUT1 protein expression in ESCs. Interestingly, the increase in GLUT1 was much higher in the hESCs (up to 10-fold by d 9 culture), compared with our murine ESC cultures. This could be attributed to two possibilities. First, the time course for our in vitro decidualization of hESCs is much longer than the murine system. At 3 d, the protein increases 2-fold in both systems. Second, ESCs from others rodents, such as rat and rabbit, lose their hormone dependence when cultured in vitro (34). It is possible that the in vitro hormonal dependence is different between these two species and accounts for the discrepancy in the degree of GLUT1 protein changes during uterine stromal decidualization.

Together, these data provide evidence for the existence of unique links between steroid hormones, glucose utilization, and decidualization in vitro. Our findings provide insight into the mechanism by which E2 and P4 supplementation may contribute to poor pregnancy outcome in some groups of patients at high risk for miscarriage and may ultimately lead to the development of new therapies for the treatment of infertility.

Supplementary Material

[Supplemental Data]
en.2008-1081_index.html (908B, html)

Footnotes

This work was supported by HD040810 (to K.H.M.), HD040390 (to K.H.M.), and DK070351 (to K.H.M.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online October 23, 2008

Abbreviations: Bio-ATB-BMPA, N-[2-[2-[2-[N-biotinylcaproylamino]-ethoxy]-ethoxyl]-4-[2-(trifluoromethyl)-3H-diazirin-3yl]benzoyl]-1,3-bis(mannopyranosyl-4-yloxy)-2-propylamine; db-cAMP, dibutyryl cAMP; DG, 2-deoxyglucose; dpc, day postcoitus; ER, estrogen receptor; ESC, endometrial stromal cell; E2, estrogen; GLUT, glucose transporter; hESC, human endometrial stromal cell; LPD, luteal phase defect; MPA, medroxyprogesterone-17-acetate; P4, progesterone; PR, progesterone receptor; Prp, prolactin-related protein; qPCR, quantitative RT-PCR; RPL, recurrent pregnancy loss.

References

  1. Nardo LG, Sallam HN 2006 Progesterone supplementation to prevent recurrent miscarriage and to reduce implantation failure in assisted reproduction cycles. Reprod Biomed Online 13:47–57 [DOI] [PubMed] [Google Scholar]
  2. Oates-Whitehead RM, Haas DM, Carrier JA 2003 Progestogen for preventing miscarriage. Cochrane Database Syst Rev CD003511 [DOI] [PubMed] [Google Scholar]
  3. Haas DM, Ramsey PS 2008 Progestogen for preventing miscarriage. Cochrane Database Syst Rev CD003511 [DOI] [PubMed] [Google Scholar]
  4. Carson DD, Bagchi I, Dey SK, Enders AC, Fazleabas AT, Lessey BA, Yoshinaga K 2000 Embryo implantation. Dev Biol 223:217–237 [DOI] [PubMed] [Google Scholar]
  5. Dey SK, Lim H, Das SK, Reese J, Paria BC, Daikoku T, Wang H 2004 Molecular cues to implantation. Endocr Rev 25:341–373 [DOI] [PubMed] [Google Scholar]
  6. Wang H, Dey SK 2006 Roadmap to embryo implantation: clues from mouse models. Nat Rev Genet 7:185–199 [DOI] [PubMed] [Google Scholar]
  7. Strauss JF, Barbieri RL 2004 Yen and Jaffe’s reproductive endocrinology: physiology, pathophysiology, and clinical management. 5th ed. Philadelphia: Elsevier Saunders [Google Scholar]
  8. Slayden OD, Keator CS 2007 Role of progesterone in nonhuman primate implantation. Semin Reprod Med 25:418–430 [DOI] [PubMed] [Google Scholar]
  9. Strowitzki T, Germeyer A, Popovici R, von Wolff M 2006 The human endometrium as a fertility-determining factor. Hum Reprod Update 12:617–630 [DOI] [PubMed] [Google Scholar]
  10. Gellersen B, Brosens J 2003 Cyclic AMP and progesterone receptor cross-talk in human endometrium: a decidualizing affair. J Endocrinol 178:357–372 [DOI] [PubMed] [Google Scholar]
  11. Lessey BA 2002 Implantation defects in infertile women with endometriosis. Ann NY Acad Sci 955:265–280 [DOI] [PubMed] [Google Scholar]
  12. Papanikolaou EG, Camus M, Fatemi HM, Tournaye H, Verheyen G, Van Steirteghem A, Devroey P 2006 Early pregnancy loss is significantly higher after day 3 single embryo transfer than after day 5 single blastocyst transfer in GnRH antagonist stimulated IVF cycles. Reprod Biomed Online 12:60–65 [DOI] [PubMed] [Google Scholar]
  13. Tomic D, Frech MS, Babus JK, Symonds D, Furth PA, Koos RD, Flaws JA 2007 Effects of ERα overexpression on female reproduction in mice. Reprod Toxicol 23:317–325 [DOI] [PubMed] [Google Scholar]
  14. Manolescu AR, Augustin R, Moley K, Cheeseman C 2007 A highly conserved hydrophobic motif in the exofacial vestibule of fructose transporting SLC2A proteins acts as a critical determinant of their substrate selectivity. Mol Membr Biol 24:455–463 [DOI] [PubMed] [Google Scholar]
  15. Wood IS, Trayhurn P 2003 Glucose transporters (GLUT and SGLT): expanded families of sugar transport proteins. Br J Nutr 89:3–9 [DOI] [PubMed] [Google Scholar]
  16. Joost HG, Thorens B 2001 The extended GLUT-family of sugar/polyol transport facilitators: nomenclature, sequence characteristics, and potential function of its novel members (review). Mol Membr Biol 18:247–256 [DOI] [PubMed] [Google Scholar]
  17. Medina RA, Meneses AM, Vera JC, Gúzman C, Nualart F, Rodriguez F, de los Angeles Garcia M, Kato S, Espinoza N, Monsó C, Carvajal A, Pinto M, Owen GI 2004 Differential regulation of glucose transporter expression by estrogen and progesterone in Ishikawa endometrial cancer cells. J Endocrinol 182:467–478 [DOI] [PubMed] [Google Scholar]
  18. Medina RA, Meneses AM, Vera JC, Guzman C, Nualart F, Astuya A, García MA, Kato S, Carvajal A, Pinto M, Owen GI 2003 Estrogen and progesterone up-regulate glucose transporter expression in ZR-75-1 human breast cancer cells. Endocrinology 144:4527–4535 [DOI] [PubMed] [Google Scholar]
  19. von Wolff M, Ursel S, Hahn U, Steldinger R, Strowitzki T 2003 Glucose transporter proteins (GLUT) in human endometrium: expression, regulation, and function throughout the menstrual cycle and in early pregnancy. J Clin Endocrinol Metab 88:3885–3892 [DOI] [PubMed] [Google Scholar]
  20. Guzeloglu-Kayisli O, Kayisli UA, Al-Rejjal R, Zheng W, Luleci G, Arici A 2003 Regulation of PTEN (phosphatase and tensin homolog deleted on chromosome 10) expression by estradiol and progesterone in human endometrium. J Clin Endocrinol Metab 88:5017–5026 [DOI] [PubMed] [Google Scholar]
  21. Brosens JJ, Hayashi N, White JO 1999 Progesterone receptor regulates decidual prolactin expression in differentiating human endometrial stromal cells. Endocrinology 140:4809–4820 [DOI] [PubMed] [Google Scholar]
  22. Marshall BA, Ren JM, Johnson DW, Gibbs EM, Lillquist JS, Soeller WC, Holloszy JO, Mueckler M 1993 Germline manipulation of glucose homeostasis via alteration of glucose transporter levels in skeletal muscle. J Biol Chem 268:18442–18445 [PubMed] [Google Scholar]
  23. Marshall BA, Murata H, Hresko RC, Mueckler M 1993 Domains that confer intracellular sequestration of the Glut4 glucose transporter in Xenopus oocytes. J Biol Chem 268:26193–2199 [PubMed] [Google Scholar]
  24. Carayannopoulos MO, Chi MM, Cui Y, Pingsterhaus JM, McKnight RA, Mueckler M, Devaskar SU, Moley KH 2000 GLUT8 is a glucose transporter responsible for insulin-stimulated glucose uptake in the blastocyst. Proc Natl Acad Sci USA 97:7313–7318 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Keembiyehetty C, Augustin R, Carayannopoulos MO, Steer S, Manolescu A, Cheeseman CI, Moley KH 2006 Mouse glucose transporter 9 splice variants are expressed in adult liver and kidney and are up-regulated in diabetes. Mol Endocrinol 20:686–697 [DOI] [PubMed] [Google Scholar]
  26. Calderhead DM, Kitagawa K, Tanner LI, Holman GD, Lienhard GE 1990 Insulin regulation of the two glucose transporters in 3T3-L1 adipocytes. J Biol Chem 265:13801–13808 [PubMed] [Google Scholar]
  27. Moley KH, Chi MM, Mueckler MM 1998 Maternal hyperglycemia alters glucose transport and utilization in mouse preimplantation embryos. Am J Physiol 275(1 Pt 1):E38–E47 [DOI] [PubMed] [Google Scholar]
  28. Li Q, Kannan A, Wang W, Demayo FJ, Taylor RN, Bagchi MK, Bagchi IC 2007 Bone morphogenetic protein 2 functions via a conserved signaling pathway involving Wnt4 to regulate uterine decidualization in the mouse and the human. J Biol Chem 282:31725–31732 [DOI] [PubMed] [Google Scholar]
  29. Orwig KE, Ishimura R, Muller H, Liu B, Soares MJ 1997 Identification and characterization of a mouse homolog for decidual/trophoblast prolactin-related protein. Endocrinology 138:5511–5517 [DOI] [PubMed] [Google Scholar]
  30. Clark AE, Holman GD 1990 Exofacial photolabelling of the human erythrocyte glucose transporter with an azitrifluoroethylbenzoyl-substituted bismannose. Biochem J 269:615–622 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Okuno S, Akazawa S, Yasuhi I, Kawasaki E, Matsumoto K, Yamasaki H, Matsuo H, Yamaguchi Y, Nagataki S 1995 Decreased expression of the GLUT4 glucose transporter protein in adipose tissue during pregnancy. Horm Metab Res 27:231–234 [DOI] [PubMed] [Google Scholar]
  32. Sugaya A, Sugiyama T, Yanase S, Shen XX, Minoura H, Toyoda N 2000 Expression of glucose transporter 4 mRNA in adipose tissue and skeletal muscle of ovariectomized rats treated with sex steroid hormones. Life Sci 66:641–648 [DOI] [PubMed] [Google Scholar]
  33. Brar AK, Frank GR, Kessler CA, Cedars MI, Handwerger S 1997 Progesterone-dependent decidualization of the human endometrium is mediated by cAMP. Endocrine 6:301–307 [DOI] [PubMed] [Google Scholar]
  34. Mani SK, Julian J, Lampelo S, Glasser SR 1992 Initiation and maintenance of in vitro decidualization are independent of hormonal sensitization in vivo. Biol Reprod 47:785–799 [DOI] [PubMed] [Google Scholar]
  35. Hresko RC, Kruse M, Strube M, Mueckler M 1994 Topology of the Glut 1 glucose transporter deduced from glycosylation scanning mutagenesis. J Biol Chem 269:20482–20488 [PubMed] [Google Scholar]

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[Supplemental Data]
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en.2008-1081_1.pdf (90.7KB, pdf)
en.2008-1081_2.pdf (59.7KB, pdf)

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