Peri- and Postpubertal Estrogen Exposures of Female Mice Optimize Uterine Responses Later in Life

Abstract

At birth, all female mice, including those that either lack estrogen receptor α (ERα-knockout) or that express mutated forms of ERα (AF2ERKI), have a hypoplastic uterus. However, uterine growth and development that normally accompany pubertal maturation does not occur in ERα-knockout or AF2ERKI mice, indicating ERα-mediated estrogen (E2) signaling is essential for this process. Mice that lack Cyp19 (aromatase knockout, ArKO mice), an enzyme critical for E2 synthesis, are unable to make E2 and lack pubertal uterine development. A single injection of E2 into ovariectomized adult (10 weeks old) females normally results in uterine epithelial cell proliferation; however, we observe that although ERα is present in the ArKO uterine cells, no proliferative response is seen. We assessed the impact of exposing ArKO mice to E2 during pubertal and postpubertal windows and observed that E2-exposed ArKO mice acquired growth responsiveness. Analysis of differential gene expression between unexposed ArKO samples and samples from animals exhibiting the ability to mount an E2-induced uterine growth response (wild-type [WT] or E2-exposed ArKO) revealed activation of enhancer of zeste homolog 2 (EZH2) and heart- and neural crest derivatives-expressed protein 2 (HAND2) signaling and inhibition of GLI Family Zinc Finger 1 (GLI1) responses. EZH2 and HAND2 are known to inhibit uterine growth, and GLI1 is involved in Indian hedgehog signaling, which is a positive mediator of uterine response. Finally, we show that exposure of ArKO females to dietary phytoestrogens results in their acquisition of uterine growth competence. Altogether, our findings suggest that pubertal levels of endogenous and exogenous estrogens impact biological function of uterine cells later in life via ERα-dependent mechanisms.

Keywords: aromatase; estrogen; phytoestrogen; uterine cell growth.

Figure 1.

Aromatase knockout (ArKO) mouse uterine response to E2 is impaired. (a) OVX wild-type (WT) or ArKO mice were treated with saline vehicle (V) or estrogen (E2) for 24 hours. Sections were evaluated by immunohistochemistry for the Ki67 proliferative marker. Extensive presence of positive (brown) cells was observed in WT E2-treated samples. Representative sections are shown. Note that because the ArKO uterine tissue is smaller than WT, the images were taken at higher magnification, as evidenced by the different scale bars. (b) Percentage of luminal epithelial cells that stained positive for Ki67 in samples from OVX adult V- or E2-treated WT (blue; n = 6 V, 8 E2) and ArKO (red; n = 6 V, 7 E2) mice. *P < 0.01 versus V by 2-way analysis of variance with the Fisher least significant difference posttest.

Figure 2.

Aberrant growth of peripubertal aromatase knockout (ArKO) uterine epithelial cells induced by estrogen (E2). (a) Uterine wet weights of wild-type (WT) and ArKO females on pnd31 after daily saline vehicle (V) or E2 injections on post natal day (pnd)28 to 30. (b) Histology of uterine samples of WT and ArKO females on pnd31 after daily V or E2 injections on pnd28 to 30. The sections were stained for Ki67. The right panels show a high magnification image to illustrate the increased epithelial proliferation in AKO versus WT. (c) Percentage of luminal epithelial cells that stained positive for Ki67 in samples from V- or E2-treated WT (blue; n = 5 V, 6 E2) and ArKO (red; n = 6 V, 6 E2) mice. *P < 0.01 versus V; +P < 0.01 versus WT by 2-way analysis of variance with the Fisher least significant difference posttest. h, hours.

Figure 3.

Peripubertal estrogen (E2) treatment partially restores aromatase knockout (ArKO) uterus E2-induced growth competence later in life. (a) Treatment scheme used to evaluate the impact of replacing peripubertal E2 using daily injections on post natal day (pnd) 28 to 30. Mice were OVX at 10 weeks of age and then rested for 2 weeks before treatment with saline vehicle (V ) or E2 for 24 hours. (b) Histological sections from uteri of wild-type (WT) or ArKO mice that were injected with V or E2 on pnd 28 to 30, then OVX at 10 weeks of age and treated with V or E2 for 24 hours. Sections were stained for the Ki67 proliferative marker. Note that because the ArKO uterine tissue is smaller than WT, some of the images were taken at higher magnification. (c) Percentage of luminal epithelial cells that stained positive for Ki67 in samples from pnd 28 to 30 V-treated WT (blue; n = 2 V, 2 E2) and ArKO (green; n = 3 V, 4 E2) mice or pnd 28 to 30 E2-treated WT (red; n = 2 V, 2 E2) and ArKO (purple; n = 3 V, 6 E2) mice. *P < 0.01 versus V by 2-way analysis of variance with the Fisher least significant difference posttest. h, hours; wk, week.

Figure 4.

Weekly estradiol benzoate (EB) treatment (weeks 3-10) further increases estrogen (E2)-induced growth of aromatase knockout (ArKO) uterus. (a) Treatment scheme used to evaluate the impact of repeated E2 exposure using weekly EB injections from ages 3 to 10 weeks. (b) Histological sections from uteri of wild-type (WT) or ArKO mice treated as described in a and then treated with saline vehicle (V) or E2 for 24 hours. Sections were stained for Ki67. (c) Graph of percentage of luminal epithelial cells per section with Ki67 marker counted in samples. *P < 0.01 versus V by 2-way analysis of variance with the Fisher least significant difference posttest (n = 6 WT V, 6 WT E2, 5 ArKO V, 9 ArKO E2). h, hours; wk, week.

Figure 5.

Estradiol benzoate (EB) treatment during weeks 9 to 10 is sufficient for estrogen (E2)-induced aromatase knockout (ArKO) uterine growth. (a) Treatment scheme used to evaluate the impact of recent E2 exposure using EB injections from during weeks 9 and 10. (b) Histological sections form uteri of wild-type (WT) or ArKO mice treated as described in a and then treated with saline vehicle (V) or E2 for 24 hours. Sections were stained for Ki67. Note that because the ArKO uterine tissue is smaller than WT, the images were taken at higher magnification. (c) Graph of percentage of luminal epithelial cells per section with Ki67 marker counted in samples. *P < 0.01 versus V by 2-way analysis of variance with the Fisher least significant difference posttest (n = 6 WT V, 4 WT E2, 3 ArKO V, 3 ArKO E2). h, hours; wk, week.

Figure 6.

Estradiol benzoate (EB) treatment of aromatase knockout (ArKO) (weeks 9-10) alters uterine transcriptome. (a) Heatmap cluster of log2 signal of differentially expressed genes (DEG) in saline vehicle (V)-treated OVX ArKO (responds: “No”) versus V-treated growth-competent samples (ArKO EB ovx, wild-type [WT] EB ovx and WT ovx; responds: “Yes”). Scale is log2 signal intensity (3.5-20). (b) Comparison of DEG (Bioset 1) to Bioset 2 from mouse embryonic fibroblasts (MEF) overexpressing HAND2, NKX2-5 GATA5, and MEF2C versus empty vector indicated positive correlation of differential expression of 171 genes.

Figure 7.

Exposure to phytoestrogen-containing diet partially restores aromatase knockout (ArKO) uterus estrogen (E2)-induced growth. (a) Treatment scheme used to evaluate the impact exposure to diet containing phytoestrogens from weeks 3 to 10. (b) Histological sections from uteri of wild-type (WT) or ArKO mice treated as described in a and then treated with saline vehicle (V) or E2 for 24 hours. Sections were stained for Ki67. Note that because the ArKO uterine tissue is smaller than WT, the images were taken at higher magnification. (c) Graph of percentage of luminal epithelial cells per section with Ki67 marker counted in samples. *P < 0.01 versus V by 2-way analysis of variance with the Fisher least significant difference posttest (n = 4 WT V, 4 WT E2, 4 ArKO V, 5 ArKO E2). h, hours; wk, week.

Figure 8.

Mechanism by which the endometrium acquires competence for estrogen (E2)-induced growth. The uterus undergoes postpubertal maturation as a result of repeated exposure to E2 with each estrous cycle. Following maturation, epithelial cell proliferation following acute E2 treatment demonstrates competency. E2 working through estrogen receptor α (ERα) decreases HAND2 and EZH2 signaling and increases GLI1 signaling. Without pubertal E2 response, as in the aromatase knockout (ArKO) or ERα knockout (ERαKO), epithelial competency is not observed. Providing exogenous pubertal E2 to ArKO mice, via estradiol benzoate (EB) dosing or through dietary E2, can induce endometrial competency. Exposure to endocrine-disrupting chemicals (EDCs) during the window of pubertal maturation might impact the process. Abbreviations: EZH2, enhancer of zeste 2 polycomb repressive complex 2; GLI1, GLI family zinc finger 1; HAND2, heart- and neural crest derivatives expressed 2.