Stromal progesterone receptors mediate induction of Indian Hedgehog (IHH) in uterine epithelium and its downstream targets in uterine stroma

Abstract

Uterine receptivity to embryo implantation depends on appropriate progesterone (P4) and estrogen stimulation. P4 rapidly stimulates production of the morphogen Indian hedgehog (IHH) in murine uterine epithelium as well as downstream molecules in the hedgehog pathway such as Patched homolog 1 (PTCH1) and nuclear receptor subfamily 2, group F, member 2 (NR2F2) in uterine stroma. Studies using IHH-null mice indicate that IHH is obligatory for the normal P4 response in the uterus. To determine whether IHH induction in uterine epithelium is mediated through P4 receptor (PR) in epithelium (E) and/or stroma (S), we produced tissue recombinants using uteri from neonatal PR knockout (ko) mice and wild-type (wt) mice containing PR in S and/or E or lacking PR altogether using a tissue recombinant methodology and assessed their response to P4. In tissue recombinants containing wt-S (wt-S + wt-E and wt-S + ko-E), P4 induced Ihh mRNA expression at 6 h that was 6-fold greater than in oil-treated controls (P < 0.05; n = 6) in both types of tissue recombinants despite the absence of epithelial PR in wt-S + ko-E grafts. Conversely, Ihh mRNA expression was unaffected by P4 in ko-S + ko-E and ko-S + wt-E grafts despite epithelial PR expression in the latter. Nr2f2 and Ptch1 mRNA expression was similar in that it was stimulated by P4 only in recombinants containing stromal PR. These results indicate that stromal PR is both necessary and sufficient for P4 stimulation of epithelial IHH as well as downstream events such as PTCH1 and NR2F2 increases in stroma.

Figure 1

Relative roles of stromal and epithelial PR in the induction of epithelial Ihh mRNA in response to P4. A, In uterine tissue recombinations containing wt-S (wt-S + wt-E and wt-S + ko-E), P4 induced a robust increase in Ihh mRNA compared with these tissue recombinations in oil-treated hosts. In contrast, uterine tissue recombinations lacking wt-S (ko-S + wt-E and ko-S + ko-E) did not have an increase in Ihh mRNA over levels seen in oil-treated controls, despite the presence of epithelial PR in ko-S + wt-E tissue recombinations. Thus, Ihh mRNA is regulated by P4 signaling through stromal PR. The Ihh mRNA values were normalized against Cdh1 mRNA to control for varying amounts of epithelium present in different tissue recombinants and allow valid comparisons between groups. B, In neonatal pups, P4 treatment induced a 170-fold increase in Ihh mRNA in uterine epithelium compared with oil-treated controls. Ihh mRNA in uterine stroma was low and not induced by P4. Ihh mRNA in uterine stroma is expressed relative to Ihh mRNA of oil-treated uterine epithelium. Cdh1 expression in uterine epithelium was unchanged by P4. Bars with different superscripts are significantly different at P < 0.01. Each data point is based on n ≥ 6, and Ihh mRNA expression in various groups was compared by one-way ANOVA followed by Tukey-Kramer’s multiple-comparison tests.

Figure 2

Expression of Ptch1 (A) and Nr2f2 (B) mRNA in uterine tissue recombinants in response to P4. In uterine tissue recombinations containing wt-S (wt-S + wt-E and wt-S + ko-E), P4 induced a robust increase in Ptch1 mRNA compared with those tissue recombinations in oil-treated hosts. In contrast, uterine tissue recombinations lacking wt-S (ko-S + wt-E and ko-S + ko-E) showed Ptch1 mRNA comparable to oil-treated controls, despite epithelial PR in ko-S + wt-E tissue recombinations. Expression of Nr2f2 mRNA in uterine tissue recombinants in response to P4 was similar to that seen with Ptch1 mRNA, in that P4 induced Nr2f2 mRNA only when stromal PR was present, and epithelial PR status did not affect Nr2f2 mRNA expression. Bars with different superscripts are significantly different at P < 0.01. All Ptch1 and Nr2f2 mRNA values were normalized against Vim mRNA to control for varying amounts of stroma present in different tissue recombinants and allow valid comparisons between groups.

Figure 3

Localization of PR expression in tissue recombinants. Uterine tissue recombinations of wt-S + wt-E showed PR immunostaining in both stromal (S) and epithelial (E) compartments (A). Conversely, in wt-S + ko-E tissue recombinations (C), PR immunostaining was confined to the stroma, whereas epithelium lacked PR. In ko-S + wt-E (B) tissue recombinations, PR immunostaining was detected only in epithelium. In ko-S + ko-E (D) tissue recombinations, PR immunostaining was undetectable. Scale bar, 10 μm.

Figure 4

Immunohistochemical localization of NR2F2 expression in uterus in response to P4. In wt-S + wt-E uterine tissue recombinations (panel A), P4 induced increased stromal (S) NR2F2 expression. NR2F2 was not expressed in the epithelium (E). In wt-S + ko-E tissue recombinations (panel C), P4 induced similar increases in stromal NR2F2 expression despite absence of epithelial PR. Conversely, in ko-S + wt-E (panel B) or ko-S + ko-E (panel D) tissue recombinations, NR2F2 expression was not stimulated by P4, despite PR expression in epithelial cells of ko-S + wt-E grafts. A negative control of a wt-S + ko-E tissue recombination processed without primary antibody did not show staining (panel E). Scale bar, 10 μm.

Figure 5

Model for P4 induction of epithelial IHH and downstream stromal molecules such as PTCH1 and NR2F2 in uterus. P4 binds to PR in stromal cells, inducing an increase in epithelial IHH through an as yet undefined stromal-epithelial interaction. IHH production in epithelium results in PTCH1 and NR2F2 expression in stroma. This mechanism of P4 response represents the first known reciprocal communication loop known in uterus, in which communication from the stroma in response to P4 leads to the production of an epithelial molecule, and that epithelial molecule then acts back on stroma to produce changes in expression of other molecules downstream of the IHH response, such as PTCH1 and NR2F2.