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. 2017 Aug 21;7(1):8377.
doi: 10.1038/s41598-017-07728-1.

Juxtacrine Activity of Estrogen Receptor α in Uterine Stromal Cells is Necessary for Estrogen-Induced Epithelial Cell Proliferation

Wipawee Winuthayanon  1 , Sydney L Lierz  2 , Karena C Delarosa  3 , Skylar R Sampels  3 , Lauren J Donoghue  2 , Sylvia C Hewitt  2 , Kenneth S Korach  2
Affiliations

Juxtacrine Activity of Estrogen Receptor α in Uterine Stromal Cells is Necessary for Estrogen-Induced Epithelial Cell Proliferation

Wipawee Winuthayanon et al. Sci Rep. .

Abstract

Aberrant regulation of uterine cell growth can lead to endometrial cancer and infertility. To understand the molecular mechanisms of estrogen-induced uterine cell growth, we removed the estrogen receptor α (Esr1) from mouse uterine stromal cells, where the embryo is implanted during pregnancy. Without ESR1 in neighboring stroma cells, epithelial cells that line the inside of the uterus are unable to grow due to a lack of growth factors secreted from adjacent stromal cells. Moreover, loss of stromal ESR1 caused mice to deliver fewer pups due in part due to inability of some embryos to implant in the uterus, indicating that stromal ESR1 is crucial for uterine cell growth and pregnancy.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Selective deletion of ESR1 anti-mesometrial mouse uterine stromal cells. ESR1 deletion was confirmed using immunohistochemical (IHC) staining of ESR1 in whole uterine sections; mesometrium (M) and anti-mesometrium (AM), in ovarian intact adult (12-week-old) Esr1 f/− and Amhr2 Cre/+; Esr1 f/− females. LE = luminal epithelial cells, GE = glandular epithelial cells, and Myo = myometrium. Representative images shown.
Figure 2
Figure 2
Uterine response to E2 treatment (24 h) in the absence of anti-mesometrial stromal ESR1. Adult (8–12-week-old) Esr1 f/− and Amhr2 Cre/+; Esr1 f/− females were ovariectomized and treated with vehicle or E2 for 24 h. (A) Uterine wet weight after 24 h of E2 treatment. *p < 0.05; significant difference between vehicle and E2 treated samples within genotype. N = 3 mice/genotype/treatment. (B) Uterine epithelial cell proliferation determined by Ki67 IHC staining in Esr1 f/− and Amhr2 Cre/+; Esr1 f/− uteri. (C) Higher magnification of Amhr2 Cre/+; Esr1 f/− treated with E2 for 24 h. Uterine sections were stained with Ki67 and ESR1 antibodies in adjacent sections. Note that epithelial cell proliferation, as indicated by the appearance of Ki67, is primarily observed in the M where ESR1 is expressed in the adjacent stromal cells. (D) Percentage of Ki67-positive cells of total luminal epithelial cells in M vs. AM regions. *p < 0.05; significant difference between vehicle and E2 treated samples within genotype and region. # p < 0.05; significant difference between Esr1 f/− and Amhr2 Cre/+; Esr1 f/− uteri after E2 treatment in the AM region, unpaired t-test. N = 4–8 mice/genotype/treatment. All graphs represent mean ± SEM. M = Mesometrium, AM = Anti-mesometrium. Representative images shown.
Figure 3
Figure 3
Cell proliferation-related uterine transcripts and protein in ovariectomized 8–12-week-old Esr1 f/− and Amhr2 Cre/+; Esr1 f/− females treated with E2 for 6 h. (A) Real-time PCR was performed and the relative expression values of Igf1, Mad2l1, Cdkn1a, Cebpb, Klf4, Mcm2, Mcm4, and Klf15 were normalized to Rpl7. *, ***p < 0.05, 0.001; significant difference between vehicle and E2 treated samples within genotype. # p < 0.05; significant difference between E2 treated samples between genotype; unpaired t-test. (B) CEBPB protein expression after 6 h of E2 treatment in Esr1 f/− and Amhr2 Cre/+; Esr1 f/− uteri using IHC analysis. All graphs represent mean ± SEM. N = 3–5 mice/genotype/treatment. M = Mesometrium, AM = Anti-mesometrium. Representative images shown.
Figure 4
Figure 4
E2-induced progesterone receptor (PGR) expression in the uterus. Adult (8–12-week-old) female mice were ovariectomized and treated with vehicle or E2 for 24 h. (A) Top panel: Cross-sections of the whole uteri were stained with PGR antibodies. Bottom panels: PGR expression pattern in the mesometrial (M) vs. anti-mesometrial (AM) poles in Esr1 f/− and Amhr2 Cre/+; Esr1 f/− uteri. Representative images shown. (B) Relative signal intensities of nuclear (Nuc) and cytosolic (Cyto) compartments in the uterine luminal epithelial cells of Esr1 f/− and Amhr2 Cre/+; Esr1 f/− uteri after vehicle and E2 treatment for 24 h. (C) Percentage of PGR-positive cells of total stromal cells in M vs. AM regions. *p < 0.05; significant difference between vehicle and E2 treated samples within genotype and region. All graphs represent mean ± SEM. N = 3 mice/genotype/treatment.
Figure 5
Figure 5
Fertility study of Amhr2 Cre/+; Esr1 f/− and Esr1 f/− mice. (A) Number of total pups per dam during 6-month breeding trial with wild-type males. Each data point represents an individual dam. ****p < 0.0001; unpaired t-test. N = 9–10 mice/genotype. (B) Ovulatory responses of 3–5-week-old females to gonadotropins indicated by total number of ovulated oocytes/mice. N = 6 mice/genotype. (C) Total number of 4.5 dpc implantation sites in 8–12-week-old female mice after natural mating. ****p < 0.0001; unpaired t-test. N = 3–4 mice/genotype. (D) Hematoxylin and eosin staining of implantation sites (4.5 dpc) in uterine cross sections. N = 3–4 mice/genotype. E = embryo, M = Mesometrium, AM = Anti-mesometrium. (E) Expression of implantation markers (Lif, Ihh, Wnt5a, and Hbegf) and E2-target transcripts (Muc1, Ltf, and Clca3) at 3.5 dpc in Esr1 f/− and Amhr2 Cre/+; Esr1 f/− 8–12-week-old female mice. *p < 0.05; unpaired t-test. N = 5–6 mice/genotype. All graphs represent mean ± SEM. Representative images shown.
Figure 6
Figure 6
Proliferation of the uterine stromal cells after a series of E2 and P4 treatments (E+Pe) to mimic the hormonal profile during implantation and at 4.5 dpc. (A) Uterine wet weights of Esr1 f/− and Amhr2 Cre/+; Esr1 f/− 8–12-week-old females mice treated with E + Poil or E + Pe. *p < 0.05; significant difference between E+Pe treated Esr1 f/− vs. Amhr2 Cre/+; Esr1 f/− females. (B) EdU incorporation assay of Esr1 f/− and Amhr2 Cre/+; Esr1 f/− female mice that were treated with E+Poil or E+Pe. Cells with green signal represent EdU positive (DNA synthesis) cells. Cells with blue signal represent the nuclei stained with Hoescht. (C) Real-time PCR analysis of Lif. Values were normalized to Rpl7. **p < 0.01; significant difference between E+Poil and E+Pe treated samples within genotype. All graphs represent mean ± SEM. (D) CEBPB IHC staining of Esr1 f/− and Amhr2 Cre/+; Esr1 f/− female mice treated with E+Poil or E+Pe. Arrowheads indicate glandular epithelial cells. N = 4–6 mice/genotype/treatment. M = Mesometrium, AM = Anti-mesometrium. (E) Expression of CEBPB and ESR1 proteins of implantation sites from Esr1 f/− and Amhr2 Cre/+; Esr1 f/− uterine cross sections at 4.5 dpc using IHC analysis. Representative images shown. N = 3–4 mice/genotype. E = embryo.
Figure 7
Figure 7
Impaired decidual response to artificial stimulation in the absence of stromal ESR1 in the anti-mesometrium. Artificial stimulation was used to induce decidual response in adult (8–12-week-old) Esr1 f/− and Amhr2 Cre/+; Esr1 f/− female mice (details for the treatment regime are described in Methods). (A) Gross morphology of uteri showing un-stimulated vs. stimulated uterine horns. Data below indicate the numbers of animals responding to the stimulation in Esr1 f/− (6/10) or Amhr2 Cre/+; Esr1 f/− (2/9) animals. Non-responsive Amhr2 Cre/+; Esr1 f/− uteri (7/9) are also shown. (B) Uterine weight increased in the stimulated horns compared to un-stimulated horns (N = 9–10 animals/group). *p < 0.05, significant difference, unpaired t-test. (C) Transcript levels of decidual markers and cell-cycle regulators (Bmp2, Prl8a2, Ccnb1, and Cdc2a) in Esr1 f/− (N = 6 of 10 animals) vs. Amhr2 Cre/+; Esr1 f/− (N = 2 of 9 animals) uteri that responded to artificial stimulation. # p < 0.05, significant difference between un-stimulated and stimulated horns within genotype, unpaired t-test. All graphs represent mean ± SEM. (D) Uterine cross-section of Esr1 f/− and Amhr2 Cre/+; Esr1 f/− mice after artificial stimulation. Images illustrate ESR1 IHC and EdU incorporation assay in un-stimulated and stimulated horns. Green indicates cells in S-phase of DNA synthesis. Blue represents Hoescht stained nuclei.
Figure 8
Figure 8
Expression of ESR1, PGR, Ki67 and marker genes for decidualization at 5.5 dpc and decidual cell proliferation at 7.5 dpc in the absence of anti-mesometrial stromal ESR1 in 8–12-week-old females. IHC analysis of (A) ESR1 and PGR and (B) Ki67 in Esr1 f/− and Amhr2 Cre/+; Esr1 f/− uteri collected at 5.5 and 7.5 dpc. N = 3–4 mice/genotype. (C) Transcript levels of decidualization markers in the uteri collected from decidual zones at 5.5 dpc, including Egfr, Ptgs2, Pgr, Wnt4, Bmp2, and Prl8a2. Graphs represent mean ± SEM. N = 3–4 mice/genotype. Representative images shown.
Figure 9
Figure 9
Resorption sites and the transcripts of angiogenesis markers at 10.5 dpc in Esr1 f/− (N = 3) and Amhr2 Cre/+; Esr1 f/− (N = 8) adult (8–12-week-old) female mice. (A) Percentage of resorption sites at 10.5 dpc in Esr1 f/− vs. Amhr2 Cre/+; Esr1 f/uteri. (B) Transcript levels of angiogenic factors (Vegfa, Vegfb, Vegfc, Flt1, Kdr, Flt4, Angpt2, and Adm), anti-angiogenic factor (Thbs1), and gap junction protein alpha 1 (Gja1) of implantation sites at 10.5 dpc. *p < 0.05; unpaired t-test. All graphs represent mean ± SEM.
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