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. 1998 Dec 22;95(26):15677-82.
doi: 10.1073/pnas.95.26.15677.

Generation and reproductive phenotypes of mice lacking estrogen receptor beta

J H Krege  1 , J B Hodgin, J F Couse, E Enmark, M Warner, J F Mahler, M Sar, K S Korach, J A Gustafsson, O Smithies
Affiliations

Generation and reproductive phenotypes of mice lacking estrogen receptor beta

J H Krege et al. Proc Natl Acad Sci U S A. .

Abstract

Estrogens influence the differentiation and maintenance of reproductive tissues and affect lipid metabolism and bone remodeling. Two estrogen receptors (ERs) have been identified to date, ERalpha and ERbeta. We previously generated and studied knockout mice lacking estrogen receptor alpha and reported severe reproductive and behavioral phenotypes including complete infertility of both male and female mice and absence of breast tissue development. Here we describe the generation of mice lacking estrogen receptor beta (ERbeta -/-) by insertion of a neomycin resistance gene into exon 3 of the coding gene by using homologous recombination in embryonic stem cells. Mice lacking this receptor develop normally and are indistinguishable grossly and histologically as young adults from their littermates. RNA analysis and immunocytochemistry show that tissues from ERbeta -/- mice lack normal ERbeta RNA and protein. Breeding experiments with young, sexually mature females show that they are fertile and exhibit normal sexual behavior, but have fewer and smaller litters than wild-type mice. Superovulation experiments indicate that this reduction in fertility is the result of reduced ovarian efficiency. The mutant females have normal breast development and lactate normally. Young, sexually mature male mice show no overt abnormalities and reproduce normally. Older mutant males display signs of prostate and bladder hyperplasia. Our results indicate that ERbeta is essential for normal ovulation efficiency but is not essential for female or male sexual differentiation, fertility, or lactation. Future experiments are required to determine the role of ERbeta in bone and cardiovascular homeostasis.

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Figures

Figure 1
Figure 1
Targeted disruption of the ERβ gene. (Top) The unmodified genomic locus showing exons 2–4. The probe used for Southern blots is labeled above exon 2. The small arrows indicate the positions of primers. Restriction enzyme sites are EcoRI, BglII, and PstI, designated by E, B, and P. (Middle) The targeting plasmid with the left (1.3-kb) and right (7.4-kb) regions of homology. The Neo insertion site is indicated. NotI (N) was used to linearize the plasmid. Homologous recombination is indicated by the large Xs. (B) shows the BglII lost in the construct. (Bottom) The targeted allele with the Neo gene inserted into exon 3. Note the additional EcoRI site introduced during the targeting. Mouse genomic DNA is shown as a thick line, the inserted Neo sequences as a thinner line, and the plasmid vector as a zig-zag line.
Figure 2
Figure 2
RT-PCR for ERβ mRNA in +/+ and −/− tissues. (A) Gel electrophoresis of the products. The arrowheads indicate the bands corresponding to full-length ERβ mRNA and the β-actin control mRNA. The marker is labeled m. A lane loaded with PCR product in the absence of reverse transcriptase is labeled “−RT”. (B) Diagrammatic representation of the RT-PCR products (see text) from wild-type and mutant mRNA (ERβ-KO1, ERβ-KO2, and ERβ-KO3) derived by using the primers indicated by the black arrows. The top line (Protein) shows the domains of the protein corresponding to the exons 1–9. A/B, N-terminal domain; C, DNA-binding domain; D, hinge domain; and E/F, ligand-binding domain. The site of the Neo gene insertion is indicated by the open arrow.
Figure 3
Figure 3
Histology and ERβ immunocytochemistry in wild-type and ERβ −/− ovary. Hematoxylin-stained sections of adult wild-type (A) and ERβ −/− (B) ovary at ×40 magnification; the arrows indicate mature follicles. (C) Immunocytochemistry of wild-type ovary with an antiserum against ERβ; o, oocyte within the follicle. (D) Immunocytochemistry of wild-type ovary with the antiserum preabsorbed with immunogenic ERβ peptide. (E) Immunocytochemistry of adult ERβ −/− ovary with the ERβ antiserum. Histologic sections of a representative ovary from an immature wild-type female (F) and from an immature ERβ −/− female (G), both after superovulation; several unruptured preovulatory follicles in G are indicated by solid arrows. Corpora lutea (cl) are indicated in F and G by open arrows.
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