GRB2 regulation of essential signaling pathways in the endometrium is critical for implantation and decidualization

Abstract

Over 75% of failed pregnancies involve implantation defects. Growth factor receptor-bound protein 2 (GRB2) is an adaptor protein involved in signal transduction and cell communication. Here we show that the expression of GRB2 protein is lower in endometrium of infertile women with endometriosis compared to controls. Our mouse endometriosis model revealed that endometriosis development results to GRB2 loss in the eutopic endometrium. To understand the role of GRB2 in the uterus, we generated mice with conditional ablation of Grb2 in the Pgr positive cells (Grb2^d/d). Grb2^d/d mice were infertile due to implantation failure. Although ovarian functions were normal, Grb2^d/d mice had a non-receptive endometrium due to progesterone resistance and dysregulation of steroid hormone and FOXA2 signaling pathways. Furthermore, our results were supported by findings of GRB2 attenuation in primary human endometrial stromal cells from women with endometriosis. Our results demonstrate that GRB2 is critical for endometrial receptivity and decidualization.

Fig. 1. The expression of GRB2 was decreased in the endometrium of women with endometriosis and women without endometriosis.

a Representative image of GRB2 immunohistochemistry in the endometrium from infertile women with endometriosis (n = 18), infertile women without endometriosis (n = 13) compared to controls (n = 19) at mild-secretory stage. The quantification of GRB2 immunohistochemistry in the stroma and epithelial cells of endometrium from women with endometriosis, women without endometriosis and control. The results represent the mean ± SEM. ***p = 0.0002, **p = 0.0033, ***p = 0.0007, **p = 0.0086 by Ordinary one-way ANOVA test. b, c The ablation of GRB2 in the uterus were confirmed by Western blot analysis (n = 3 per genotype) and immunohistochemistry in the uteri of Grb2^f/f and Grb2^d/d mice at GD 3.5 (n = 5 per genotype). d Representative fluorescence photomicrographs and average total number of endometriotic sites in Grb2^f/f and Grb2^d/d mice by induced endometriosis based on mT/mG mice (n = 5 per genotype). Endometriotic lesions were visualized by GFP expression in the outside of uterus. White arrow indicates endometriotic lesions. The results represent the mean ± SEM by two-tailed unpaired t-test. e Immunohistochemistry GRB2 analysis from uteri of the endometrium from 1 month and 3 months after endometriosis induction and Sham mice at GD 3.5 (n = 6 per genotype and time point). Semi-quantitative analysis of GRB2 levels in the epithelium and stroma in the stroma and epithelial cells endometrium from 3 months after endometriosis induction and Sham mice at GD 3.5. The results represent the mean ± SEM. *p = 0.0334, *p = 0.0234 and *p = 0.0384, *p = 0.0445, ***p < 0.0001 by Ordinary one-way ANOVA test. Source data are provided in the Source Data file.

Fig. 2. Implantation defect and altered PTGS2 expression in Grb2^d/d mice at GD 4.5.

a Representative image of GRB2 immunohistochemistry in the uterine tissue from GD 0.5 to GD 7.5 (n = 5 per time point). b Visualizations of implantation sites by Chicago Sky Blue 6B dye in Grb2^f/f and Grb2^d/d uteri at GD 4.5 (n = 6 per genotype) after intravenous injection of Chicago Sky Blue 6B dye. c Hematoxylin and eosin analysis of implantation sites from uteri of Grb2^f/f (i–iii) and Grb2^d/d (iv–ix) mice at GD 4.5. d Immunohistochemistry PTGS2 analysis from uteri of Grb2^f/f (i and ii) and Grb2^d/d (iii–vi) mice at GD 4.5 (n = 3).

Fig. 3. Dysregulation of FOXO1 and PGR at the implantation sites of Grb2^d/d mice at GD 4.5.

a Immunohistochemistry analysis of FOXO1 from the uteri of Grb2^f/f (i and ii) and Grb2^d/d (iii–vi) mice at GD 4.5. b Semi-quantitative analysis of FOXO1 levels in the epithelium of Grb2^f/f and Grb2^d/d mice at GD 4.5 (n = 5 per group). The results represent the mean ± SEM. ***p < 0.0001, ***p < 0.0001, and **p = 0.0033 by Ordinary one-way ANOVA test. c Immunohistochemistry analysis of PGR from the uteri of Grb2^f/f (i and ii) and Grb2^d/d (iii–vi) mice at GD 4.5. d Semi-quantitative analysis of PGR levels in the epithelium and stroma of Grb2^f/f and Grb2^d/d mice at GD 4.5 (n = 5 per group). The results represent the mean ± SEM. ***p < 0.0001, *p = 0.0125, and *p = 0.0163 by Ordinary one-way ANOVA test. Source data are provided in the Source Data file.

Fig. 4. Decidualization defects in Grb2^d/d mice and hESCs.

a A decrease of the stimulated/control uterine weight ratio in Grb2^f/f and Grb2^d/d mice at decidualization day 5 (n = 5 for Grb2^f/f and n = 4 for Grb2^d/d mice). The results represent the mean ± SEM. ***p = 0.0001 by two-tailed unpaired t-test. b Hematoxylin and eosin (H&E) staining in control and stimulated horn of Grb2^f/f and Grb2^d/d mice at decidualization day 5. c RT-qPCR analysis for the expression of decidualization marker genes, Bmp2, Fst, Fkbp5, and Wnt4, in the uteri of Grb2^f/f and Grb2^d/d mice (n = 4 per genotype). The results represent the mean ± SEM. ***p < 0.0001, ***p < 0.0001, ***p < 0.0001, ***p < 0.0001, *p = 0.0435, **p = 0.0045, **p = 0.0034, **p = 0.0042 by Ordinary one-way ANOVA test. d The expression of GRB2 in hESCs from control women without (Ctrl) and with (Eosis) endometriosis (n = 22 per group). The expression of GRB2, IGFBP1, and PRL levels in hESCs and after silencing with siRNA in decidualized HESCs. Each treatment was performed with at three biological replicates (n = 3 per group and time point). The results represent the mean ± SEM. ***p = 0.0001, **p = 0.0085, *p = 0.0200, **p = 0.0035, ***p = 0.0004, and ***p < 0.0001, by Ordinary one-way ANOVA test. Source data are provided in the Source Data file.

Fig. 5. Loss of Grb2 in uterine results in non-receptive endometrium due to progesterone resistance.

a Immunohistochemistry of Ki67 analysis in the uteri of Grb2^f/f and Grb2^d/d mice at GD 3.5 (n = 5 per genotype). The results represent the mean ± SEM. ***p < 0.0001 and ***p < 0.0001 by two-tailed unpaired t-test. b RT-qPCR analysis for E2 target genes, Clca3, Ltf, and Muc-1, in the uteri of Grb2^f/f and Grb2^d/d mice at GD 3.5 (n = 4 per genotype). The results represent the mean ± SEM. **p = 0.0063, *p = 0.0221, and ***p = 0.0006 by two-tailed unpaired t-test. c Immunohistochemistry of ESR1 analysis in the uteri of Grb2^f/f and Grb2^d/d mice at GD 3.5 (n = 5 per genotype). The results represent the mean ± SEM. ** p = 0.0014 by two-tailed unpaired t-test. d RT-qPCR analysis for P4 target genes, Il13ra2, Fst, Areg, and Lrp2, in the uteri of Grb2^f/f and Grb2^d/d mice at GD 3.5 (n = 4 per genotype). The results represent the mean ± SEM. **p = 0.0015, ***p = 0.0009, **p = 0.0019 and ***p < 0.0001 by two-tailed unpaired t-test. e Immunohistochemistry of PGR analysis in the uteri of Grb2^f/f and Grb2^d/d mice at GD 3.5 (n = 5 per genotype). The results represent the mean ± SEM. ***p < 0.0001 by two-tailed unpaired t-test. f Immunohistochemistry of stromal maker protein COUP-TFII and g Vimentin in the uteri of Grb2^f/f and Grb2^d/d mice at GD 3.5 (n = 5 per genotype). The results represent the mean ± SEM. *** p < 0.0001, and *** p < 0.0001 by two-tailed unpaired t-test. Source data are provided in the Source Data file.

Fig. 6. Transcriptome profile of the uteri in Grb2^f/f and Grb2^d/d mice at GD 3.5.

a GO terms of the upregulated genes in the uteri of Grb2^d/d mice compared with that of Grb2^f/f mice from RNA-seq data. b Heatmap of E2-related genes and P4-related genes in the uterus of Grb2^f/f and Grb2^d/d mice at GD 3.5. c Heatmap of uterine EGFR-related genes and d RT-qPCR and immunohistochemistry analysis of EGFR in the uteri of Grb2^f/f and Grb2^d/d mice at GD 3.5 (n = 4 per genotype). The results represent the mean ± SEM. *p = 0.0221 by two-tailed unpaired t-test. e Immunohistochemistry analysis of EGFR and pERK1/2 in the uteri of Grb2^f/f and Grb2^d/d mice at GD 4.5. f Semi-quantitative analysis of EGFR and pERK1/2 levels in the uteri of Grb2^f/f and Grb2^d/d mice at GD 4.5 (n = 5 per genotype). The results represent the mean ± SEM. ***p < 0.0001, ***p < 0.0001, *p = 0.0300, *p = 0.0455, ***p < 0.0001, and ***p < 0.0001 by Ordinary one-way ANOVA test. Source data are provided in the Source Data file.

Fig. 7. ERK signaling is critical for implantation and decidualization.

a The number of implantation sites were analyzed by 3D reconstruction of implantations from mice treated with U0126 at GD 5.5, GD 7.5, and GD 9.5 (n = 5 per treatment). b Representative image of implantation site of mice treated with vehicle and U0126 at GD 9.5. c Mice treated with U0126 shows a decreased number of implantation sites compared with mice treated with vehicle at GD 9.5 (n = 5 per treatment). The results represent the mean ± SEM. **p = 0.0088 by two-tailed unpaired t-test. Source data are provided in the Source Data file.

Fig. 8. A decrease of FOXA2 expression in the uteri in Grb2^d/d mice at GD 3.5.

a The number of grands in the uteri of Grb2^f/f and Grb2^d/d mice at GD 3.5 (n = 6). The results represent the mean ± SEM. ***p < 0.0001 by two-tailed unpaired t-test. b Immunohistochemistry analysis of FOXA2 in the uteri of Grb2^f/f and Grb2^d/d mice at GD 3.5 (n = 5 per treatment). The results represent the mean ± SEM. ***p < 0.0001 by two-tailed unpaired t-test. c Venn diagram of identifying genes that overlapped in RNA-Seq data between Foxa2^d/d and Grb2^d/d mice at GD 3.5. d A heatmap of differentially expressed genes in uterus of control, Foxa2^d/d and Grb2^d/d mice at GD 3.5 from RNA-Seq data (n = 4 per genotype). e Heatmap of Grb2 and Foxa2-related genes in the uteri of Foxa2^d/d and Grb2^d/d mice at GD 3.5 (n = 4 per genotype). Source data are provided in the Source Data file.