GLUT4 in Mouse Endometrial Epithelium: Roles in Embryonic Development and Implantation

Abstract

GLUT4 is involved in rapid glucose uptake among various kinds of cells to contribute to glucose homeostasis. Prior data have reported that aberrant glucose metabolism by GLUT4 dysfunction in the uterus could be responsible for infertility and increased miscarriage. However, the expression and precise functions of GLUT4 in the endometrium under physiological conditions remain unknown or controversial. In this study, we observed that GLUT4 exhibits a spatiotemporal expression in mouse uterus on pregnant days 1-4; its expression especially increased on pregnant day 4 during the window of implantation. We also determined that estrogen, in conjunction with progesterone, promotes the expression of GLUT4 in the endometrial epithelium in vivo or in vitro. GLUT4 is an important transporter that mediates glucose transport in endometrial epithelial cells (EECs) in vitro or in vivo. In vitro, glucose uptake decreased in mouse EECs when the cells were treated with GLUT4 small interfering RNA (siRNA). In vivo, the injection of GLUT4-siRNA into one side of the mouse uterine horns resulted in an increased glucose concentration in the uterine fluid on pregnant day 4, although it was still lower than in blood, and impaired endometrial receptivity by inhibiting pinopode formation and the expressions of leukemia inhibitory factor (LIF) and integrin ανβ3, finally affecting embryonic development and implantation. Overall, the obtained results indicate that GLUT4 in the endometrial epithelium affects embryo development by altering glucose concentration in the uterine fluid. It can also affect implantation by impairing endometrial receptivity due to dysfunction of GLUT4.

Keywords: embryonic development; endometrial epithelium; glucose transporter 4; implantation; uterine receptivity.

FIGURE 1

| GLUT4 expression in the mouse endometrium on pregnant days 1–4. Mouse uterine tissues were collected on days 1–4 of pregnancy. One side of the uterine horns was fixed in 4% paraformaldehyde for paraffin blocks and the other side was stored at −80°C for Western blotting analysis, n = 10 per group. (A) Immunohistochemistry (IHC) staining for GLUT4 expression in the mouse endometrium. Le, luminal epithelium; Str, stromal tissue. Arrows indicate the luminal epithelium. GLUT4 expression in the mouse duodenum for the positive control, without the primary antibody for the negative control. Scale bar, 10 μm. (B) Western blotting analysis for GLUT4 expression in the mouse endometrium on pregnant days 1–4. Relative density analysis of GLUT4 by Image Lab 3.0 software (n = 3) normalized by β-actin. Different superscript letters (a, b, c) denote significant differences among groups (P < 0.05); the same superscript letters mean no significant difference (P > 0.05).

FIGURE 2

Estrogen and progesterone regulate the expression of GLUT4 in the endometrial epithelium. Mouse uteri were collected at 24 h after the last injection. One side of the uterine horns was fixed in 4% paraformaldehyde for paraffin blocks and the other side was stored at −80°C for Western blotting analysis, n = 10 per group. (A) Immunohistochemistry (IHC) staining for GLUT4 expression in ovariectomized (OVX) mouse endometrium 2 days after hormone treatments. Control (oil), E2 (0.2 μg/day estrogen), P4 (4 mg/day progesterone), and E2 + P4 (0.2 μg/day E2 + 4 mg/day P4). Negative control is without the primary antibody (shown at the bottom of the left-hand corner). Le, luminal epithelium; Str, stromal tissue. n = 10 per group. Scale bar, 10 μm. (B) Western blotting analysis for GLUT4 expression in OVX mouse uterine tissues (n = 3, normalized by β-actin). (C) Immunofluorescence staining for GLUT4 expression in mouse endometrial epithelial cells (EECs) 48 h after hormone treatments: control (solvent control), E2 (10^–8 mol/L), P4 (10^{− 6} mol/L), and E2 + P4 (combination of 10^–8 mol/L E2 and 10^{− 6} mol/L P4). Negative control is without the primary antibody. The experiment was performed at least three times. Images were taken at × 40. Scale bar, 10 μm. (D) Quantitative analysis of immunofluorescence staining using Image-Pro Plus 6.0 software. (E) Western blotting analysis for GLUT4 expression in mouse EECs. The cells were harvested 48 h after hormone treatments. Relative density analysis of the GLUT4 proteins by Image Lab 3.0 software (n = 3, normalized by β-actin). ns, not significant. *P < 0.05, **P < 0.01, ***P < 0.01 vs. control; ^#P < 0.05 vs. the E2 group.

FIGURE 3

Effect of GLUT4 on glucose uptake in mouse endometrial epithelial cells (EECs) following GLUT4-siRNA transfection. (A) The 2-NBDG (green) signals in mouse EECs shown by fluorescent scope. The cells in five visual fields per group were counted and photographed. The intensity of 2-NBDG in mouse EECs was analyzed by Image-Pro Plus 6.0 software. ***P < 0.001. Scale bar, 500 μm. (B) The uptake of 2-NBDG in mouse EECs was detected by microcapillary flow cytometry (Millipore, United States) at the FITC channel. The abscissa is the fluorescence intensity and the ordinate is the cell number. The curve shift to the left indicated a dropped glucose uptake in mouse EECs. (C) The expressions of p-AMPK and AMPK in mouse EECs were detected by Western blotting. The ratio of p-AMPK/total AMPK reflects the state of intracellular energy demand in mouse EECs (the ratio increases as the intracellular energy demand increases). The relative densities of the p-AMPK and AMPK proteins were normalized by β-actin. *P < 0.05 vs. control. The experiment was performed at least three times.

FIGURE 4

Effect of GLUT4 on glucose concentration in the uterine fluid and endometrial receptivity following GLUT4-siRNA transfection. (A) Uterine fluid was collected on pregnant day 4 using in vivo uterine perfusion and glucose concentration in the uterine fluid was measured by HPLC. The glucose concentration in the GLUT4-siRNA side was obviously higher than that in the control side (4.12 ± 0.57 vs. 2.01 ± 0.29 mM, ^∗P < 0.05 vs. control, n = 12), but was still lower than that in blood (6.7 ± 0.72 mM). (B) Pinopode formation in the luminal epithelium was observed by SEM on pregnant day 4. Arrows indicate pinopodes. Scale bar, 30 μm. (C) Western blotting analysis for leukemia inhibitory factor (LIF) expression on pregnant day 4 following GLUT4-siRNA transfection. Relative density analysis of the LIF protein by Image Lab 3.0 software (n = 3, normalized by β-actin). (D) Western blotting for integrin ανβ3 expression following GLUT4-siRNA transfection (n = 3, normalized by β-actin). *P < 0.05 vs. control.

FIGURE 5

Effect of GLUT4 on embryonic development and implantation. (A) On pregnant day 4, embryonic development was observed. Arrows indicate abnormal blastocysts. The number of normal blastocysts in the GLUT4-siRNA side was obviously decreased compared to that in the control side (3.5 ± 0.6 vs. 9.3 ± 0.8, ***P < 0.001 vs. control, n = 12). (B) On pregnant day 5, the implantation site was observed. The number of implantation sites in the GLUT4-siRNA side was significantly decreased compared to that in the control side (2.3 ± 1.2 vs. 7 ± 0.6, *P < 0.05 vs. control, n = 12). (C) Embryonic implantation rates on pregnant day 5 of embryo transfer. Eight blastocysts from donor mice were transferred into one uterine horn of each recipient mouse. The implantation rate in the GLUT4-siRNA side was significantly lower than that in the control side (0.19 ± 0.07% vs. 0.41 ± 0.07%, *P < 0.05 vs. control, n = 12).