Uterine Foxl2 regulates the adherence of the Trophectoderm cells to the endometrial epithelium

Abstract Background Forkhead Transcription Factor L2 (FOXL2) is a member of the forkhead family with important roles in reproduction. Recent studies showed that FOXL2 is expressed in human and bovine endometrium and that its levels fluctuate during pregnancy. In this study, we aimed at evaluating the expression and function of FOXL2 in embryo implantation. Methods Mouse uteri at different days of pregnancy were isolated and analyzed for the expression and localization of FOXL2. A lentiviral strategy was further employed to either knockdown or overexpress FOXL2 in non-receptive human endometrial AN3-CA cells and in receptive Ishikawa cells, respectively. These genetically modified cells were compared to cells infected with a control lentivirus to determine the function of FOXL2 in trophectoderm cells adherence to Endometrial Epithelium was associated with the expression of genes known to be involved in acquisition of uterine receptivity. Results We report that FOXL2 is expressed in both, the luminal epithelium and the myometrium of the mouse uterus and that its expression declines prior to implantation. We found that endometrial cells expressing low FOXL2 levels, either endogenous or genetically manipulated, were associated with a higher attachment rate of mouse blastocysts or human Jeg3 spheroids and mouse blastocysts. In accordance, low-FOXL2 levels were associated with changes in the expression level of components of the Wnt/Fzd and apoptotic pathways, both of which are involved in uterine receptivity. Furthermore, FOXL2 expression was inversely correlated with G-protein signaling protein 2 (Rgs2) and cytokine expression. Conclusions These results suggest that FOXL2 interferes with embryo attachment. Better understanding of the function of FOXL2 in the uterus would possibly suggest novel strategies for treatment of infertility attributed to repeated implantation failure

Additional file 1: of Uterine Foxl2 regulates the adherence of the Trophectoderm cells to the endometrial epithelium

Figure S1. Embryos attachment to endometrial cells. Mouse blastocysts co-incubated with endometrial cell lines for 48 h (A). Attached embryos that stayed on the plate after the plates were washed and shaken (B). Thin arrows indicate attached embryos, thick arrow indicate unattached embryos. (C) The localization of osteopentin, an adhesion molecule, between the embryo and the endometrial cells. Osteopentin-green, DAPI-blue. (TIFF 2025 kb

Expression and Regulation of Corticotropin-Releasing Factor Receptor Type 2 beta in Developing and Mature Mouse Skeletal Muscle

Corticotropin-releasing factor receptor type 2 (CRFR2) is highly expressed in skeletal muscle (SM) tissue where it is suggested to inhibit interactions between insulin signaling pathway components affecting whole-body glucose homeostasis. However, little is known about factors regulating SM CRFR2 expression. Here, we demonstrate the exclusive expression of CRFR2, and not CRFR1, in mature SM tissue using RT-PCR and ribonuclease protection assays and report a differential expression of CRF receptors during C2C12 myogenic differentiation. Whereas C2C12 myoblasts exclusively express CRFR1, the C2C12 myotubes solely express CRFR2. Using cAMP luciferase assays and calcium mobilization measurements, we further demonstrate the functionality of these differentially expressed receptors. Using luciferase reporter assays we show a differential activation of CRFR promoters during myogenic differentiation. Transfections with different fragments of the 5′-flanking region of the mCRFR2β gene fused to a luciferase reporter gene show a promoter-dependent expression of the reporter gene and reveal the importance of the myocyte enhancer factor 2 consensus sequence located at the 3′-proximal region of CRFR2β promoter. Furthermore, we demonstrate that CRFR2 gene transcription in the mature mouse is stimulated by both high-fat diet and chronic variable stress conditions. Performing a whole-genome expression microarray analysis of SM tissues obtained from CRFR2-null mice or wild-type littermates revealed a robust reduction in retinol-binding protein 4 expression levels, an adipokine whose serum levels are elevated in insulin-resistant states. In correlation with the SM CRFR2β levels, the SM retinol-binding protein 4 levels were also elevated in mice subjected to high-fat diet and chronic variable stress conditions. The current findings further position the SM CRFR2 pathways as a relevant physiological system that may affect the known reciprocal relationship between psychological and physiological challenges and the metabolic syndrome

Additional file 3: of Uterine Foxl2 regulates the adherence of the Trophectoderm cells to the endometrial epithelium

Figure S2. FOXL2 localization in the ovary. FOXL2 expressed by granulosa cells of early follicles declines at later stages of folliculogenesis. Blue = DNA staining (DAPI), RED = FOXL2. EA-Early antral, GF-Graffian Folicle. (TIFF 2025 kb