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Estrogen receptor ? exon 3-deleted mouse: The importance of non-ERE pathways in ER? signaling.

ABSTRACT: In 1998, an estrogen receptor ? (ER?) knockout (KO) mouse was created by interrupting the gene at the DNA binding domain (DBD) with a neocassette. The mutant females were subfertile and there were abnormalities in the brain, prostate, lung, colon, and immune system. In 2008, another ER? mutant mouse was generated by deleting ER? exon 3 which encodes the first zinc finger in the DBD. The female mice of this strain were unable to ovulate but were otherwise normal. The differences in the phenotypes of the two KO strains, have led to questions about the physiological function of ER?. In the present study, we created an ER? exon 3-deleted mouse (ER?-?ex3) and confirmed that the only observable defect was anovulation. Despite the two in-frame stop codons introduced by splicing between exons 2 and 4, an ER? protein was expressed in nuclei of prostate epithelial cells. Using two different anti-ER? antibodies, we showed that an in-frame ligand binding domain and C terminus were present in the ER?-?ex3 protein. Moreover, with nuclear extracts from ER?-?ex3 prostates, there was an ER?-dependent retardation of migration of activator protein-1 response elements in EMSA. Unlike the original knockout mouse, expression of Ki67, androgen receptor, and Dachshund-1 in prostate epithelium was not altered in the ER?-?ex3 mouse. We conclude that very little of ER? transcriptional activity depends on binding to classical estrogen response elements (EREs).

SUBMITTER: Maneix L

PROVIDER: S-EPMC4413313 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Estrogen receptor β exon 3-deleted mouse: The importance of non-ERE pathways in ERβ signaling.

Maneix Laure L Antonson Per P Humire Patricia P Rochel-Maia Sabrina S Castañeda Jessica J Omoto Yoko Y Kim Hyun-Jin HJ Warner Margaret M Gustafsson Jan-Åke JÅ

Proceedings of the National Academy of Sciences of the United States of America 20150406 16

In 1998, an estrogen receptor β (ERβ) knockout (KO) mouse was created by interrupting the gene at the DNA binding domain (DBD) with a neocassette. The mutant females were subfertile and there were abnormalities in the brain, prostate, lung, colon, and immune system. In 2008, another ERβ mutant mouse was generated by deleting ERβ exon 3 which encodes the first zinc finger in the DBD. The female mice of this strain were unable to ovulate but were otherwise normal. The differences in the phenotypes ...[more]

PMID: 25848008

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Project description:The estrogen receptor beta (ERβ) is physiologically essential for reproductive biology and is implicated in various diseases. However, despite more than 20 years of intensive research on ERβ, there are still uncertainties about its distribution in tissues and cellular expression. Several studies show contrasts between mRNA and protein levels, and the use of knockout strategies revealed that many commercially available antibodies gave false-positive expression results. Recently, a specific monoclonal antibody against human ERβ (PPZ0506) showed cross-reactivity with rodents and was optimized for the detection of rat ERβ. Herein, we established an immunohistochemical detection protocol for ERβ protein in mouse tissue. Staining was optimized on murine ovaries, as granulosa cells are known to strongly express ERβ. The staining results were confirmed by western blot analysis and RT-PCR. To obtain accurate and reliable staining results, different staining conditions were tested in paraffin-embedded tissues. Different pitfalls were encountered in immunohistochemical detection. Strong heat-induced epitope retrieval (HIER) and appropriate antibody dilution were required to visualize specific nuclear expression of ERβ. Finally, the specificity of the antibody was confirmed by using ovaries from Esr2-depleted mice. However, in some animals, strong (non-specific) background staining appeared. These signals could not be significantly alleviated with commercially available additional blocking solutions and are most likely due to estrus-dependent expression of endogenous immunoglobulins. In summary, our study showed that the antibody PPZ0506, originally directed against human ERβ, is also suitable for reliable detection of murine ERβ. An established staining protocol mitigated ambiguities regarding the expression and distribution of ERβ in different tissues and will contribute to an improved understanding of its role and functions in murine tissues in the future.

Dataset Information

Estrogen receptor ? exon 3-deleted mouse: The importance of non-ERE pathways in ER? signaling.

Publications

Estrogen receptor β exon 3-deleted mouse: The importance of non-ERE pathways in ERβ signaling.

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