Adherens junction proteins in the hamster uterus: their contributions to the success of implantation

Abstract

The adherens junction (AJ) is important for maintaining uterine structural integrity, composition of the luminal environment, and initiation of implantation by virtue of its properties of cell-cell recognition, adhesion, and establishment of cell polarity and permeability barriers. In this study, we investigated the uterine changes of AJ components E-cadherin, beta-catenin, and alpha-catenin at their mRNA and protein levels, together with the cellular distribution of meprinbeta, phospho-beta-catenin, and active beta-catenin proteins, in hamsters that show only ovarian progesterone-dependent uterine receptivity and implantation. By in situ hybridization and immunofluorescence, we have demonstrated that uterine epithelial cells expressed three of these AJ proteins and their mRNAs prior to and during the initial phase of implantation. Immunofluorescence study showed no change in epithelial expression patterns of uterine AJ proteins from Days 1 to 5 of pregnancy. With advancement of the implantation process, AJ components were primarily expressed in cells of the secondary decidual zone (SDZ), but not in the primary decidual zone (PDZ). In contrast, we noted strong expression of beta-catenin and alpha-catenin proteins in the PDZ, but not in the SDZ, of mice. Taken together, these results suggest that AJ proteins contribute to uterine barrier functions by cell-cell adhesion to ensure protection of the embryo. In addition, cleavage of E-cadherin by meprinbeta might contribute to weakening uterine epithelial cell-cell contact for blastocyst implantation. We also report that the nuclear localization of active beta-catenin from Day 4 onward in hamsters implies that beta-catenin/Wnt-signal transduction is activated in the uterus during implantation and decidualization.

FIG. 1.

In situ hybridization of Cdh1, Ctnnb1, and Ctnna1 mRNAs in early pregnant hamster and mouse uteri. A) Cross sections of hamster uteri from Days 1, 4, and 6 demonstrated cell-specific localization of Cdh1, Ctnnb1, and Ctnna1 mRNAs. B) Cross sections of implant sites from mouse Days 5 and 6 demonstrated cell-specific localization of Ctnnb1 and Ctnna1 mRNAs. Dark field photomicrographs were representative of a single experiment using uterine sections from two to three animals in the same slide. No positive signals were observed when sections were hybridized with the sense probes. bl, blastocyst; cm, circular muscle; em, embryo; ge, glandular epithelium; le, luminal epithelium; lm, longitudinal muscle; PDZ, primary decidual zone; s, stroma; SDZ, secondary decidual zone.

FIG. 2.

Immunolocalization (FITC; green) of E-cadherin, total β-catenin, and α-catenin in early pregnant hamster and mouse uteri. A) Uterine cross sections from Days 1 to 6 of pregnancy in hamsters showed cell-specific localization of E-cadherin, total β-catenin, and α-catenin proteins. B) Uterine cross sections from mouse Days 5 and 6 implant sites showed cell-specific localization of total β-catenin and α-catenin proteins. The nuclei were stained with DAPI (blue). The photomicrographs (original magnification ×100) shown are representative of at least five to seven independent experiments. No specific immune staining was observed in negative control sections. To show the specificity of total β-catenin and α-catenin immunolocalization, negative control pictures from Day 4 uterus in Figure 2A and Day 5 implant site in Figure 2B were presented, because both antibodies were raised in the same species (rabbit). Insets (original magnification ×400) showed immunolocalization of proteins in the epithelium (Day 2) or myometrium (Day 4). bl, blastocyst; em, embryo; ge, glandular epithelium; le, luminal epithelium; PDZ, primary decidual zone; s, stroma; SDZ, secondary decidual zone.

FIG. 3.

E-cadherin (FITC; green) and total-β-catenin (TRITC; red) double labeling in the same uterine sections on Days 4 to 6 of pregnancy in hamsters. The nuclei were stained for DAPI (blue). The photomicrographs shown are representative of at least three independent experiments. bl, blastocyst; em, embryo; le, luminal epithelium; PDZ, primary decidual zone; s, stroma; SDZ, secondary decidual zone.

FIG. 4.

Immunolocalization (FITC; green) of meprinβ in cross sections of pregnant hamster uteri from Days 1 to 5. The nuclei were stained for DAPI (blue). The photomicrographs (original magnification ×200) shown are representative of at least three to four independent experiments with sections from three animals. bl, blastocyst; ge, glandular epithelium; le, luminal epithelium; s, stroma.

FIG. 5.

Immunolocalization (FITC; green) of phospho-β-catenin and active β-catenin in uterine cross sections from Days 1 to 4 and implantation sites from Days 5 and 6 of pregnancy in hamsters. The nuclei were stained with DAPI (blue). The photomicrographs shown are representative of at least three to four independent experiments, with sections from more than one animal in the same slides. No or low staining was observed in negative control (ctrl) sections. bl, blastocyst; em, embryo; ge, glandular epithelium; le, luminal epithelium; PDZ, primary decidual zone; s, stroma; SDZ, secondary decidual zone.

FIG. 6.

Immunolocalization (FITC; green) of phospho-β-catenin and active β-catenin in uterine cross sections from implantation sites of Days 5 and 6 of pregnancy in mice. The nuclei were stained with DAPI (blue). The photomicrographs shown are representative of at least two to three independent experiments, with sections from more than one animal in the same slides. No or low staining was observed in negative control sections. bl, blastocyst; em, embryo; ge, glandular epithelium; le, luminal epithelium; PDZ, primary decidual zone s, stroma; SDZ, secondary decidual zone.