Uterine Wnt/beta-catenin signaling is required for implantation

Abstract

Successful implantation relies on precisely orchestrated and reciprocal signaling between the implanting blastocyst and the receptive uterus. We have examined the role of the Wnt/beta-catenin signaling pathway during the process of implantation and demonstrate that this pathway is activated during two distinct stages. Wnt/beta-catenin signaling is first transiently activated in circular smooth muscle forming a banding pattern of activity within the uterus on early day 4. Subsequently, activation is restricted to the luminal epithelium at the prospective site of implantation. Activation at both sites requires the presence of the blastocyst. Furthermore, inhibition of Wnt/beta-catenin signaling interferes with the process of implantation. Our results demonstrate that the Wnt/beta-catenin signaling pathway plays a central role in coordinating uterus-embryo interactions required for implantation.

Fig. 1.

The Wnt/β-catenin signaling pathway is activated in the circular smooth muscle cells of the myometrium. (A) Whole-mount β-galactosidase staining of a uterus from a nonpregnant transgenic female. (B) Whole-mount β-galactosidase staining of a uterus from a TCF/Lef-lacZ transgenic female at mid-day 4. (C) Whole-mount β-galactosidase staining of a uterus from a TCF/Lef-lacZ transgenic female on the evening of day 4. (D and E) Sagittal (D) and transverse (E) sections of the uterus in C showing activity in the circular smooth muscle cells (csm).

Fig. 2.

Activation of the Wnt/β-catenin signaling pathway in the luminal epithelium requires the presence of the embryo and is estrogen-dependent. (A) Whole-mount β-galactosidase staining of a TCF/Lef-lacZ transgenic uterus at 1200 hours on day 5 showing restricted activity within the uterus. (B) Transverse section from a uterus at 1200 hours on day 5 showing activation of the reporter gene in the luminal epithelium (le) adjacent to the blastocyst (bl) only on the antimesometrial side (ams) of the uterus and not on the mesometrial side (ms). (C) Higher magnification of B (×50). (D) Transverse section from a TCF/Lef-lacZ uterus at 1600 hours on day 5 demonstrating that activity has spread throughout the luminal epithelium by this stage. (E) Higher magnification of D (×200). (F) Whole-mount β-galactosidase staining of a uterus from a TCF/Lef-lacZ transgenic female mated with a vasectomized male. (G) Wholemount β-galactosidase staining of a uterus from a TCF/Lef-lacZ transgenic female where the junction between the oviduct and the uterus was tightly ligated on one side. Speckled staining in uterine horn corresponds to endometrial glands. (H and I) Uterus from a pregnant TCF/Lef-lacZ transgenic female in which both ovaries were removed on the morning of day 4, before the estrogen surge, and injected with progesterone alone (H) or progesterone and estrogen (I).

Fig. 3.

Wnt proteins known to activate the canonical Wnt pathway can selectively activate Wnt/β-catenin signaling in the uterus. (A) Whole-mount β-galactosidase staining of a TCF/Lef-lacZ transgenic uterus injected with Wnt5a-conditioned media in both uterine horns. (B) Whole-mount β-galactosidase staining of a TCF/Lef-lacZ transgenic uterus injected with Wnt7a-conditioned media in both uterine horns. (C) Whole-mount β-galactosidase staining of a TCF/Lef-lacZ transgenic uterus injected with Wnt7a-conditioned media only in the right uterine horn. (D) A Cytodex-3 bead (bd) is approximately the same size as a wild-type blastocyst (bl). (E) A Cytodex-3 bead coated with Wnt7a-expressing cells. (F) Whole-mount β-galactosidase staining of a TCF/Lef-lacZ transgenic uterus injected with Wnt5a-coated Cytodex-3 beads. (G) Whole-mount β-galactosidase staining of a TCF/Lef-lacZ transgenic uterus injected with Wnt7a-coated Cytodex-3 beads.

Fig. 4.

Activation of the Wnt/β-catenin signaling pathway in the uterus is required for proper blastocyst implantation. (A) Uterine horn on day 6 in which blastocysts along with BSA had been transferred on day 4. (B) Uterine horn on day 6 in which blastocysts along with sFRP1 had been transferred on day 4. (C) Uterine horn on day 6 in which blastocysts along with sFRP2 had been transferred on day 4. (D) Uterus on day 6 in which sFRP2-conditioned media along with embryoid bodies expressing sFRP2 had been injected in the right uterine horn on the morning of day 4. The left horn was not injected and served as a control. (E) Embryos dissected from a uterine horn that had been injected with sFRP2-conditioned media along with embryoid bodies expressing sFRP2 (Right) and embryos from the same uterus from the control uterine horn (Left).

Fig. 5.

Model for blastocyst-induced Wnt/β-catenin signaling in the uterus. On the morning of day 4, the blastocyst emits a signal that results either directly or indirectly in the activation of the Wnt/β-catenin signaling pathway in discrete groups of cells within the circular smooth muscle, which form bands along the uterus. On late day 4 to early day 5, the blastocyst activates the Wnt/β-catenin signaling pathway in the luminal epithelium of the uterus. Activation of the Wnt/β-catenin signaling pathway results in the modulation of target genes in the uterus, which facilitates implantation.