Estrogen Receptor Null Mice - Endocrine Reviews

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June, 1999 ESTROGEN RECEPTOR NULL MICE 369 FIG. 3. Histology of uterine and vaginal tissue of wild-type, �ERKO, and �ERKO females. Cross-sections of uterine tissue from (a) wild-type, (b) �ERKO, and (c) �ERKO adult females illustrating the presence of all three anatomical tissue compartments in the uteri of the wild-type and ERKO mice (33�; inset, 132�). The representative wild-type uterine section illustrates a normal myometrium (My), endometrial stroma (St), and epithelium (Ep) (inset). The representative �ERKO uterine section illustrates the characteristic hypoplasia of each compartment, a slightly disorganized endometrial stroma, and a lack of estrogenization of the luminal and glandular epithelium (inset). Note the dramatically smaller diameter of the �ERKO uterus, as indicated by the ability to fit the whole transverse section of the tissue in the field of view. The representative �ERKO uterine section is indistinguishable from that of the wild-type, including the presence of estrogen-stimulated luminal epithelium (inset). Below are cross-sections of vaginal tissue from representative (d) wild-type, (e) �ERKO, and (f) �ERKO female mice (66�). The representative wild-type vaginal section illustrates a normal stroma (St) and hypertrophied epithelium (Ep) showing estrogen-induced stratification and cornification. In contrast, these estrogen actions are lacking in the �ERKO vagina. Once again, the tissue of the �ERKO is indistinguishable from the wild-type control. Scale bar � 1 �m. teresting in light of the increased levels of estradiol found in the serum of adult �ERKO females (Table 2). However, Lindzey et al. (154) demonstrated that the concurrently elevated ovary-derived androgens in the �ERKO female (Table 2) do provide for some maintenance of uterine weight, which can be further reduced upon ovariectomy. We recently reported that ER� mRNA is barely detectable in the adult mouse uterus, including those from �ERKO mice (93), making it unlikely that ER� could provide a compensatory role in the �ERKO uterus. Numerous immunohistochemical studies for ER� and the apparent loss of estrogen sensitivity in the �ERKO uterus indicate that the classical ER� is the predominant form responsible for mediating estrogen actions in the mouse uterus. The response of the ovariectomized rodent uterus to estradiol has been well documented and is often described as biphasic, with the initial phase consisting of effects that become apparent within the first 6hofasingle estrogen treatment (reviewed in Ref. 155). These include metabolic responses in the form of increased water imbibition, vascular permeability and hyperemia, prostaglandin release, glucose metabolism, and eosinophil infiltration (155). A series of biosynthetic responses are also characteristic of the first phase and include increased RNA polymerase and chromatin activity, lipid and protein synthesis, and increased glucose-6-phosphate dehydrogenase (155). As several of the above processes continue, they are accompanied by dramatic increases in RNA and DNA synthesis, mitosis, and cellular hyperplasia and hypertrophy that peak 24–72 h after exposure (155). Initial studies suggested that this biphasic pattern may be the result of at least two types of acceptor sites for estradiol-receptor complexes in the uterus (156). However, the uteri of �ERKO females fail to exhibit components of both phases after estrogen treatment, providing strong evidence for the requirement of ER� in the full response (46, 123). In brief, when treated with 40 �g E 2 or diethylstilbestrol (DES) per kg body weight for three consecutive days, wild-type mice exhibited the expected 3- to 4-fold increase in uterine
370 COUSE AND KORACH Vol. 20, No. 3 TABLE 2. Serum hormone levels in adult wild-type and �ERKO mice Hormone Gonadal steroids Wild-type (SEM) Female �ERKO (SEM) Wild-type (SEM) Male �ERKO (SEM) Estradiol (pg/ml) b 29.5 � 2.5 84.3 � 12.5 a 11.8 � 3.4 12.9 � 3.4 Progesterone (ng/ml) b 2.3 � 0.6 4.0 � 1.1 0.5 � 0.3 0.3 � 0.1 Testosterone (ng/ml) Anterior pituitary 0.4 � 0.4 3.2 � 0.6 9.3 � 4.0 16.0 � 2.3 LH (ng/ml) 0.3 � 0.04 1.7 � 0.3 a 2.4 � 1.2 3.7 � 0.7 FSH (ng/ml) 4.9 � 0.6 5.4 � 0.7 26.0 � 1.4 30.0 � 1.1 PRL (ng/ml) nd, Not determined. a t test, wild-type vs. ERKO, P � 0.001. 18.8 � 10.7 3.5 � 1.3 nd nd b These values in the female are different than those reported in Ref. 123, which were carried out on pooled sera. The values above are the means from assays on individual samples and therefore are more likely to reflect the true levels in the two genotypes. wet weight, whereas no such response was observed in the uteri of �ERKO mice (46, 157). It should be noted that this pharmacological dose of estrogen is well beyond that required to achieve a maximum response in the wild-type rodent. Nonetheless, estrogen-treated �ERKO uteri exhibited no apparent components of the initial phase of estrogen effects, including water imbibition and hyperemia. Histological analysis and [ 3 H]thymidine incorporation assays indicated a lack of significant cellular proliferation and DNA synthesis in uteri from the estrogen-treated �ERKO mice (123, 153). Interestingly, although the heterozygous females possess approximately one-half the normal complement of ER�, their uterine response to estrogens is equal to that of the wild-type females. In a similar study, wild-type and �ERKO mice treated with hydroxy-tamoxifen (1 mg/kg) produced comparable results (157), eliciting the expected estrogenic response in the wild-type and having no effect on the �ERKO uterus. These studies thereby confirm that the estrogen agonist activity of hydroxy-tamoxifen, which is somewhat unique to the mouse uterus (8), is mediated via the ER� pathway. The mitogenic and stimulatory action of estradiol in the uterus is a complex process involving increased RNA polymerase and ribosomal activity (158), resulting in the regulation of a plethora of genes. It is well accepted that the ligand-bound ER complex is not directly involved in the mediation of all responses elicited by estrogens in the uterus, but rather serves as a stimulus for a cascade of signaling pathways that act to amplify the estrogen action. However, certain genes appear to be directly regulated by the ER�estradiol complex and possess functional estrogen-responsive elements within their regulatory regions. Two such examples are the genes encoding the progesterone receptor (PR) (159, 160) and the secretory protein, lactoferrin (161). In fact, the regulation of the uterine PR and lactoferrin genes have often been used as assays for the estrogenic activity of experimental compounds. Therefore, with a similar intent, we used these estrogen markers to attest for estrogen insensitivity in the uteri of the �ERKO mouse. A single dose of estradiol, known to be effective in inducing the PR and lactoferrin genes within 24 h in uteri of wild-type mice, produced no such up-regulation in the uteri of the �ERKO mice, confirming the need for a direct action of the ER� in this mechanism (123). Interestingly, a recent report by Tibbetts et al. (162) demonstrated that the estrogen-stimulated increases in PR are localized to the stromal and myometrial compartments, whereas the increases in lactoferrin are isolated to the luminal and glandular epithelium in the mouse uterus. Therefore, disruption of the ER� gene has resulted in estrogen insensitivity in all three anatomical compartments of the uterus. However, it must be noted that constitutive levels of PR and lactoferrin mRNA are present in the �ERKO uteri, suggesting that these genes are also under the influence of pathways independent of ER�. A testimony to the complexity of estrogen action in the uterus is the finding that while estradiol up-regulates PR expression in the myometrium and stroma, it simultaneously abolishes PR levels in the luminal epithelium (162). This would indicate an inhibitory role of the estradiol-ER� complex on PR expression in this portion of the uterus. Speculating that this pathway may therefore be lacking in the �ERKO uterus, an investigation as to the source of the PR mRNA in the �ERKO uteri is warranted. 2. Changes in growth factor functions. A component of the cascade of events that lead to the obvious changes in the physiology of the adult uterus after estrogen exposure are the auto- and paracrine actions of polypeptide growth factors. Several members of the epidermal growth factor family have been suggested as possible mediators of estrogen-induced mitogenesis in the uterus. This hypothesis is based on experiments demonstrating that estradiol up-regulates the uterine levels of epidermal growth factor and its receptor (EGF, EGF-R) (163, 164), transforming growth factor-� (165), and insulin-like growth factor-I (IGF-1) (166). Furthermore, mice homozygous for a targeted disruption of the EGF-R gene exhibit a hypoplastic uterus that is significantly reduced in size (167), similar to that of the �ERKO. Experimental data indicate that treatment of ovariectomized wild-type mice with EGF mimics the early effects of estradiol and DES in terms of inducing modified cell morphology and increases in the levels of ER, DNA synthesis, phosphatidylinositol turnover, PR, and lactoferrin in the uterus (168–170). Further studies have illustrated that cotreatment with anti-EGF antibodies was able to attenuate the uterine response to estradiol, presumably due to inactivation of the EGF-signaling pathway (168). In turn, cotreatment with the estrogen antagonist ICI-164,384 was able to reduce the uterine response to EGF (169). These in vivo studies suggest a cross-talk mechanism between the EGF and ligand-independent ER- signaling pathways. The results of the animal studies have been
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Estrogen Receptor Null Mice - Endocrine Reviews

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