ERBB receptor feedback inhibitor 1 regulation of estrogen receptor activity is critical for uterine implantation in mice

Abstract

Normal endometrial function requires a balance of progesterone (P4) and estrogen (E2) effects. E2 acts to stimulate the proliferation of uterine epithelial cells, while P4 action inhibits E2-mediated proliferation of the epithelium. P4 through its cognate receptor, the P4 receptor (Pgr), has important roles in the establishment and maintenance of pregnancy. In previous studies, we have identified ERBB receptor feedback inhibitor 1 (Errfi1) as a downstream target of Pgr action in the uterus. Herein, we show that Errfi1 mRNA expression was significantly increased in the uterus after Day 2.5 of gestation. Its expression is also induced in the uterus by acute E2 treatment, and this induction is synergistically induced by chronic E2 and P4 treatment. Although it is known that conditional ablation of Errfi1 in the Pgr-positive cells (Errfi1(d/d)) results in infertility, the function of Errfi1 in reproductive biology has remained elusive. Using Errfi1(d/d) mice, we have identified Errfi1 as an important mediator of uterine implantation. Epithelial ESR1 and target genes were significantly increased in the uteri of Errfi1(d/d) mice. Our results identify a new signaling paradigm of steroid hormone regulation in female reproductive biology that adds insight into the underlying dysregulation of hormonal signaling in human reproductive disorders such as endometriosis and endometrial cancer.

FIG. 1.

The expression of Errfi1 by real-time RT-PCR and in situ hybridization in pseudopregnancy. A) The expression level of Errfi1 was measured in uteri of pseudopregnancy. Total RNA used for the RT-PCR assays was prepared from pseudopregnant uteri. The results represent the mean ± SEM of three independent RNA sets. **P < 0.01 and ***P < 0.001. B) The localization pattern of Errfi1 mRNA by in situ hybridization during early pregnancy. Nuclei are counterstained with hematoxylin. E, embryonic day; EM, embryo; LE, luminal epithelium; GE, glandular epithelium; PDZ, primary decidual zone; SDZ, secondary decidual zone; ST, stroma cells; MYO, myometrium; bar = 500 μm. Arrowheads indicate luminal and glandular epithelium.

FIG. 2.

The expression pattern of Errfi1 by E2 and P4 in the uterus. Total RNA used for the RT-PCR assays was prepared from wild-type mice that were treated with P4, E2, E2 plus P4, or vehicle (sesame oil [Veh]) for 4 h, 16 h, and 40 h. The results represent the mean ± SEM of three independent RNA sets. *P < 0.05 and **P < 0.01.

FIG. 3.

Implantation defect in Errfi1^d/d mice. Mice were euthanized at 5.5 dpc of pregnancy, and the number of implantation sites was counted by injecting Chicago Sky Blue dye. Implantation sites were not detected in the mutant uterine horns (n = 7), while normal implantation sites (mean ± SEM, 7.25 ± 0.48) were scored in the controls (n = 4). Bar = 1 cm.

FIG. 4.

An increase in expression of Esr1-regulated genes in Errfi1^d/d mice. A) Real-time RT-PCR analysis of C3, Ltf, Muc1, and Clca3 was performed on uteri of Errfi1^f/f and Errfi1^d/d mice at 3.5 dpc. The results represent the mean ± SEM of six independent mouse sets. *P < 0.05 and ***P < 0.001. B) Immunohistochemical analysis of LTF (a and b) and MUC1 (c and d) in uteri of Errfi1^f/f (a and c) and Errfi1^d/d (b and d) mice at 3.5 dpc. Bar = 200 μm.

FIG. 5.

An increase in E2 receptor activity in the uterine of Errfi1^d/d mice. A) Real-time RT-PCR analysis of Esr1 and Pgr was performed on uteri of Errfi1^f/f and Errfi1^d/d mice at 3.5 dpc. The results represent the mean ± SEM of six independent mouse sets. *P < 0.05. B) Immunohistochemical analysis of ESR1 (a and b), phospho-ESR1 (c and d), and PGR (e and f) in uteri of Errfi1^f/f and Errfi1^d/d mice at 3.5 dpc. Bar = 200 μm.