Fig 1.
Endometrial expression of FOXO1 during the preimplantation period in the uteri of pseudopregnant mice.
(A) Immunohistochemical staining for FOXO1 in endometrial cross sections of pseudopregnant (PP) mice (n = 3). (B) Immunohistochemical staining FOXO1 in sagittal sections of the implantation site (IS) and inter-implantation site (I-IS) in the afternoon of the window of receptivity (gestational day 5, GD 5)(n = 3). Blastocyst indicated by the white triangle. Scale bar, 50 μm.
Fig 2.
Ablation of Foxo1 in the endometrium results in infertility.
(A-B) Generation of a Foxo1 conditional knockout (Foxo1d/d) using the PgrCre mouse model in adult ovariectomized females treated with estrogen and progesterone to mimic day 3.5 of pregnancy. (A) Ablation of Foxo1 message was determined by qRT-PCR. Ablation of Foxo1 does not affect the expression of the progesterone (Pgr) and estrogen receptor (Esr1) genes. Data are presented as means ± SEM, n = 6. ** P<0.01. (B) Western blot analysis of Foxo1 ablation with β-actin (ACTB) as protein loading control. (C) Immunohistochemistry with a FOXO1-specific antibody of uterine cross sections in estrogen (E2) and progesterone (P4)-primed ovariectomized (OVX) mice (OVX+E2/P4) and at GD 5 (n = 3). Arrows indicate endometrial compartments as follows: LE, luminal epithelium; GE, glandular epithelium; BV, blood vessel; S, stroma; and E, embryo. IS, implantation site. Scale bars: 50 μm for top panel; 100 μm for bottom panel. (D) Intact females at 6 weeks of age were mated with fertile males. Presence of vaginal plug indicated postcoital day 0.5 (GD 0.5). Eosin and hematoxylin staining was performed on serial sagittal sections of whole uteri dissected at GD 5 (n = 3). Attachment sites in Foxo1f/f and Foxo1d/d are shown in low magnification and high magnification of dotted box area. Inner cell mass of blastocyst indicated by white triangle. LE cells indicated by grey triangles. Scale bar, 50 μm. (E) Immunohistochemical staining for PTGS1 and RNAscope in situ hybridization for Hbegf on transverse serial sections at GD 5 (n = 4). E, embryo. Scale bar, 100 μm.
Fig 3.
Ablation of Foxo1 causes continues proliferation in uterine epithelia independent of ovarian function and adenogenesis.
(A) The number of embryos flushed from Foxo1f/f and Foxo1d/d uteri at GD 3.5 (n = 5). (B) Serum P4 level from Foxo1f/f and Foxo1d/d mice at GD 3.5 (n = 5). (C) Immunohistochemical staining of FOXA2 in GD 3.5 uteri from Foxo1f/f and Foxo1d/d mice (n = 4). Scale bar, 200 μm. (D) Quantification of Foxa2 gene in GD 3.5 uteri from Foxo1f/f and Foxo1d/d mice (n = 6). (E) The number of uterine glands from Foxo1f/f and Foxo1d/d mice (n = 4) at GD 3.5. (F) Immunohistochemical staining of Ki67 in GD 3.5 uteri from Foxo1f/f and Foxo1d/d mice (n = 4). Scale bar, 100 μm. (G) H-score quantification of Ki67 in endometrial section of Foxo1f/f and Foxo1d/d mice (n = 3) at GD 3.5. Data are presented as means ± SEM. ****, P<0.0001.
Fig 4.
Ablation of Foxo1 retains the polarity of uterine epithelium in GD 4.5 pregnant mice.
(A) Immunohistochemical staining for Mucin-1 (MUC1), E-cadherin (CDH1), Connexin 26 (GJB2), ZO-1 (TJP1) and Claudin 1 (CLDN1) in the cross sections of both Foxo1f/f and Foxo1d/d murine uteri at GD 4.5. (B) Immunohistochemical staining for Caspase 3 and cleaved-Caspases 3 in the cross sections of Foxo1f/f and Foxo1d/d murine uteri at GD 5.5. M, mesometrial pole; AM, antimesometrial pole; E, embryo; ICM, inner cell mass; IS, implantation site; I-IS, inter-implantation site. Scale bar, 100 μm.
Fig 5.
FOXO1-dependent regulation of uterine genes.
(A) Heat map representation of gene expression levels determined by RNA-seq in Foxo1f/f and Foxo1d/d uteri at day 4.5 of pseudopregnancy (PPD 4.5). Differential gene expression analysis identified 631 uterine genes significantly regulated in the Foxo1d/d mice relative to Foxo1f/f. (B) Enrichment of functional annotation and (C) activities of upstream regulators in differentially expressed genes between Foxo1f/f and Foxo1d/d mouse uteri at PPD 4.5 by IPA analysis. (D) Independent samples collected for qRT-PCR validation of targets of interests in the ontology analysis of the FOXO1 gene signature. Data are presented as means ± SEM. n = 5. *, P<0.05; **, P<0.01; and ***, P<0.001.
Fig 6.
Mutually controlled balance of epithelial PGR and FOXO1 governs the WOR.
Immunohistochemical staining for FOXO1 and PGR in the cross sections of Foxo1f/f and Foxo1d/d murine uteri at GD 4.5 (A), as well as, PGR and FOXO1 in the cross sections of PgrA+/+ and PgrAOE/+ murine uteri at GD 4.5 (B). LE, luminal epithelium; S, stroma. Scale bar, 100 μm.
Fig 7.
Conserved FOXO1 and PGR balance in endometrial receptivity in human.
(A) Immunohistochemical staining for FOXO1 and PGR on endometrial sections from the functional layer of the endometrium in the proliferative, early secretory, mid-secretory, and late secretory phase of the menstrual cycle (n = 5). Scale bar, 50 μm. (B) Score for the compartmental and sub-cellular FOXO1 immunostaining across the menstrual cycle. Intensity indicated as follows: -, negative; +, weak; ++ moderate; +++, high. The slash is meant to show range given the variation in the samples (n = 5) (C) Proposed model for the temporal downregulation of Pgr in the LE of the endometrium by FOXO1 across the WOR.