Uterine WNTS modulates fibronectin binding activity required for blastocyst attachment through the WNT/CA2+ signaling pathway in mice

Abstract

Adhesion of the implanting blastocyst involves the interaction between integrin proteins expressed by trophoblast cells and components present in the basement membrane of the endometrial luminal epithelium. Although several factors regulating integrins and their adhesion to fibronectin are already known, we showed that Wnt signaling is involved in the regulation of blastocyst adhesion through the trafficking of integrins expressed by trophoblast cells. Localization of Itgα5β1 by immunofluorescence and FN-binding assays were conducted on peri-implantation blastocysts treated with either Wnt5a or Wnt7a proteins. Both Wnt5a and Wnt7a induced a translocation of Itgα5β1 at the surface of the blastocyst and an increase in FN-binding activity. We further demonstrated that uterine fluid is capable of inducing integrin translocation and this activity can be specifically inhibited by the Wnt inhibitor sFRP2. To identify the Wnt signaling pathway involved in this activity, blastocysts were incubated with inhibitors of either p38MAPK, PI3K pathway or CamKII prior to the addition of Wnts. Whereas inhibition of p38MAPK and PI3K had not effect, inhibition of CamKII reduced FN-binding activity induced by Wnts. Finally, we demonstrated that inhibition of Wnts by sFRP2 reduced the binding efficiency of the blastocyst to uterine epithelial cells. Our findings provide new insight into the mechanism that regulates integrin trafficking and FN-binding activity and identifies Wnts as a key player in blastocyst attachment to the uterine epithelium.

Keywords: Embryo implantation; Fibronectin binding; Integrins; Mouse blastocyst; Non-canonical wnt signaling; Trophectoderm; Wnt signaling; Wnt/Ca2+.

Fig. 1

Expression of Itgα5β1 in peri-implantation blastocysts treated with Wnt proteins. (A) Microscopy images of 3.5 dpc blastocysts cultured in KSOM alone or with control CM (L cells), Wnt5a CM and Wnt7a CM and stained with (total Itgα5β1) or without (surface Itgα5β1) permeabilization with anti-Itgα5β1 antibody (green). Nuclei were stained with iodure propidium (red). (B) The ratio between the green signal (Itgα5β1) and red signal (iodure propidium) was used to evaluate the total Itgα5β1 expression in 3.5 dpc blastocysts cultured for 48 h in KSOM alone, control CM, Wnt5a CM and Wnt7a CM and stained with permeabilization. (C) The fluorescent intensity of the green ratio (Itgα5β1) was used to evaluate the surface Itgα5β1 expression in 3.5 dpc blastocysts cultured for 48 h in KSOM alone, control CM, Wnt5a CM and Wnt7a CM and stained without permeabilization. * ANOVA test, P < 0.05

Fig. 2

FN-Binding assays of peri-implantation blastocysts treated with Wnt proteins. (A) Microscopy images of FN-binding assays realized on 3.5 dpc blastocysts cultured in control L cells CM, Wnt5a CM and Wnt7a CM for varying incubation times. Binding activity was visualized in green, and nuclei were stained with iodure propidium (red). (B) Fluorescent intensity of the green signal was quantified and used to evaluate the binding activity of 3.5 dpc blastocysts cultured in control L cells CM, Wnt5a CM and Wnt7a CM for varying incubation times. * 2 way ANOVA test, P < 0.05 and Šídák’s multiple comparisons test. Different letters indicate a significant difference, while the same letter indicates a non-significant difference

Fig. 3

FN-Binding assays of peri-implantation blastocysts in vitro and in vivo. (A) Microscopy images of FN-binding assays realized on 3.5 dpc blastocysts cultured in KSOM alone, uterin fluid (UF) ± sFRP2 ± Wnt7a CM. Binding activity was visualized in green and nuclei were stained with iodure propidium (red). (B) Fluorescent intensity of the green signal was quantified and used to evaluate the binding activity of 3.5 dpc blastocysts cultured in KSOM alone, uterin fluid (UF) ± sFRP2 ± Wnt7a CM. * ANOVA test, P < 0.05 and Holm-Šídák’s multiple comparisons test. (C) Microscopy images of FN-binding assays realized on 3.5 dpc blastocysts cultured in vitro in KSOM or transplanted into the uterus with KSOM ± sFRP2 ± Wnt7a CM. Binding activity was visualized in green, and nuclei were stained with iodure propidium (red). (D) Fluorescent intensity of the green signal was quantified and used to evaluate the binding activity of 3.5 dpc blastocysts in vitro in KSOM or transplanted into the uterus with KSOM ± sFRP2 ± Wnt7a CM. * ANOVA test, P < 0.05 and Holm-Šídák’s multiple comparisons test

Fig. 4

FN-Binding assays of peri-implantation blastocysts treated with Wnt proteins and different inhibitors. (A) Microscopy images of FN-binding assays realized on 3.5 dpc blastocysts cultured in control L cells CM, Wnt5a CM and Wnt7a CM with the addition of different inhibitors. Binding activity was visualized in green, and nuclei were stained with iodure propidium (red). (B) Microscopy images of FN-binding assays realized on 3.5 dpc blastocysts cultured in control L cells CM, Wnt5a CM and Wnt7a CM and with the CamKII inhibitor KN93. (C) Fluorescent intensity of the green signal was quantified and used to evaluate the binding activity of 3.5 dpc blastocysts cultured in control L cells CM, Wnt5a CM and Wnt7a CM and with the CamKII inhibitor KN93. **2way ANOVA, P < 0.01 and Dunnett’s multiple comparisons test

Fig. 5

Attachment assays between peri-implantation blastocysts and luminal epithelial uterine cells. (A) Bright-field photomicrographs of co-culture between hatched blastocysts and luminal epithelial uterine cells in KSOM alone, with sFRP2 or with sFRP2 and Wnt7a CM. (B) After 2 h of incubation, attachment of blastocysts to the epithelial cells were quantified by moving the plates and counting the number of blastocysts attached or non attached. * ANOVA test, P < 0.05 and Holm-Šídák’s multiple comparisons test