Fig 2: On-time removal of the LE is defective in LIF-replaced glandless PgrCre/+Foxa2f/f mice. Control, gland-containing LtfiCre/+Foxa2f/f and glandless PgrCre/+Foxa2f/f mice received i.p. injections of recombinant mouse LIF on GD 4 and were evaluated at 2000 hours on GD 5. a Section of implantation sites on GD 5 were stained with hematoxylin and eosin. Representative images are shown for two independent mice representing observed variation. Columns 1 and 3—Scale bar: 250 μm; Columns 2 and 4—Scale bar, 50 μm. b Immunofluorescence analysis of CDH1 in implantation sites. Areas of breached epithelium are indicted with white arrowheads. Row 1—Scale bar: 100 μm; Row 2—Scale bar: 50 μm; Row 3—Scale bar: 10 μm. c Immunofluorescence analysis of CLDN1 in implantation sites. Areas of breached epithelium are indicted with white arrowheads. Scale bar: 50 μm. AM: antimesometrial, M: mesometrial, Em: embryo, LE: luminal epithelium. All images are representative of three independent experiments

Fig 3: The uterine transcriptome is dysregulated in the implantation sites of GD 6 mice that lack endometrial glands. RNA-sequencing analysis was conducted using implantation sites from LIF-replaced glandless and control mice on GD 6 (n = 4 per genotype). a Volcano plot of all genes detected in transcriptome analysis. All data points above the red horizontal line are significant. Known gland-specific genes have red points and are labeled with gene names. b Heatmap for 53 genes (log2 FPKM values) known to be involved in decidualization that were different in GD 6 implantation sites in LIF-replaced glandless PgrCre/+Foxa2f/f mice. c Visualization of biological process GO terms associated with genes differentially expressed in LIF-replaced glandless PgrCre/+Foxa2f/f and control mice on GD 6. Color indicates the FDR P value and size indicates the frequency of the GO term in the underlying annotation database. Darker circles with a larger size represent highly significant terms that are more general

Fig 4: Stromal cell decidualization and epithelial remodeling in LIF-replaced FOXA2-deficient mice. Control, gland-containing LtfiCre/+Foxa2f/f and glandless PgrCre/+Foxa2f/f mice received i.p. injections of recombinant mouse LIF on GD 4 and were evaluated on GD 5 at 0800 hours. a Immunofluorescence analysis of PTGS2 and CK8 in implantation sites. PTGS2 is present in the stroma around the implanting embryo on the antimesometrial side of the uterus. Scale bar: 100 μm. b Immunofluorescence analysis of CDH1 in implantation sites. Scale bar: 100 μm. c Immunofluorescence analysis of CLDN1 in implantation sites. Scale bar: 100 μm. AM: antimesometrial, M: mesometrial, Em: embryo, LE: luminal epithelium, S: stroma. All images are representative of three independent experiments

Fig 5: Epithelial ESR1 deletion mice display defects in Lif expression.(A) Uterine sections of control, LER, and XER mice at GD3 1200h and GD3 1800h with staining for Hoechst (nuclei white), FOXA2 (gland marker, green), and Lif mRNA (red). Lif expression in LER glands is high in a subset of cells (b-b”, g-g”) and lower in others (c-c”, g-g”). Lif expression increases from GD3 1200h to GD3 1800h in LER glands while XER glands express undetectable levels of Lif at both time points. Scale bar, 100μm. (B) Quantitative analysis of the amount of Lif per gland volume in control, XER and LER mice at GD3 1200h and GD3 1800h. n=9 regions each from 3 mice per genotype per stage were analyzed. Data in (B) analyzed using Kruskal-Wallis test with Dunn’s multiple comparisons. (*) = p<0.05. (C) Uterine horn of XER mouse intraluminally injected with 1μg recombinant Lif into the right horn on GD3 1200h and dissected on GD4 1200h following blue dye injection. Asterisks indicate implantation rescue sites, and the total number of embryos found in each horn are shown.