Estrogen Receptor Null Mice - Endocrine Reviews

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June, 1999 ESTROGEN RECEPTOR NULL MICE 365 et al. (88) have demonstrated colocalization of ER� and ER� to select regions of the rat forebrain. C. Generation of the ER null mice The field of estrogen action has been and continues to be a dynamic one with broadening scope. Although estradiol is often called the female sex steroid, the list of tissues and organs in which estrogen actions appear to be critical to physiological function continues to expand, in both sexes. The crucial actions of estradiol in the female reproductive tract, breast, and hypothalamic-pituitary axis have been well described. However, we now know of significant functions fulfilled by estrogens in male reproduction. Furthermore, the effects of estrogens in the physiology, maintenance, and overall health of the cardiovascular, central nervous, bone, immune, skin, and adipogenesis systems have been an area of continued research. And finally, the role that estrogen and normal and variant ERs may play in carcinogenesis, especially in the breast and female reproductive tract, continues to receive great attention. The rationale for generating mice that possess no functional ER is multifaceted, but in its most simple terms, was founded on the classical ablation experiments of the early part of this century. In 1900, Knauer (105) described the ability of ovarian grafts to prevent uterine atrophy in the castrated guinea pig. Five years later, Marshall and Jolly (106) described the capacity of ovarian extracts to induce estrus when administered to ovariectomized dogs. Similar protocols were later elegantly employed by Jost (107) to substantiate the endocrine function of the testis and the importance of testosterone in sex determination. These studies were critical to establishing the following basic criteria required to verify an endocrine role for a particular organ or tissue: 1) removal or destruction of the synthesizing organ should result in predictable symptoms presumed to be related to the absence of the hormone; 2) administration of material prepared from the removed organ should relieve these symptoms; and 3) the hormone should be present in and extractable from both the organ and blood (108). In the spirit of these earlier studies, the later part of this century has witnessed the marriage of two relatively new methodologies to introduce a modern version of the ablation experiment. The combination of in vitro culture of mouse embryonic stem cells and targeted homologous recombination has generated a tool that allows for the precise disruption or knockout of a particular gene of study and the passage of this mutation to offspring. Although one can debate whether this new technique is more or less invasive than the previous surgical methods, it is obvious that the classic “ablation” experiment has been elevated to a molecular level. The function of a specific hormone can now be studied rather than the function of a whole endocrine organ that may produce multiple secretions. Furthermore, this new technology allows for the study of a particular cellular component, such as a receptor, that is intrinsic to one or more tissues. Such studies were previously impossible or relied on the use of chemical antagonists, which introduced their own inherent limitations. Additionally, gene targeting provides for in vivo methods to study the roles of a particular receptor throughout the life of the animal, including the early development stages. The current tools that allow for the generation of transgenic mice have already made significant contributions to our knowledge of particular genes, especially those involved in development and reproduction (reviewed in Refs. 109 and 110). At the time the ERKO mouse was envisioned, the ER� was the only form of ER known to exist. Furthermore, there were no reports of ER mutations in normal tissue that resulted in estrogen insensitivity in humans or laboratory animals. This was in contrast to the descriptions of syndromes of receptorbased insensitivity to androgens (37, 38) and thyroid hormones (40). Therefore, investigators were inclined to conclude that mutations that resulted in insufficient estrogen synthesis or resistance to the hormone at the level of the target organ were lethal at the earliest developmental stages (111, 112). This view was strengthened by reports of the detection of ER� mRNA in both human (113) and mouse (114) oocytes as well as in mouse blastocysts (115). Accordingly, the concept of generating a mouse devoid of ER� was initially met with skepticism but was pursued as a collaborative effort between our laboratory and that of Dr. Oliver Smithies of the University of North Carolina at Chapel Hill. If disruption of the mouse ER� gene did prove lethal, a model to study the precise time and locations of critical ER-mediated actions during early development would be available. However, providing the animal was viable, an in vivo model of estrogen insensitivity would now be accessible for continued study. Six years after the initial description of the �ERKO mouse, we now take for granted that disruption of the ER� gene proved not to be lethal, but rather the animal develops normally and exhibits a life span comparable to its wild-type litter mates (46). However, as will be elaborated in detail in this review, the adult �ERKO mice exhibit several abnormalities and deficiencies, most notable of which are the phenotypic syndromes that result in infertility in both sexes. Since this time, additional discoveries have been made to enhance the utility of the �ERKO model. Soon after the generation of the �ERKO mouse, Smith et al. (116) reported the first and only known case of clinical estrogen insensitivity due to an inactivating mutation of the ER� gene in a human patient. Later came multiple descriptions of aromatase and subsequent estradiol deficiency in humans (117–120). And finally, a second collaborative effort resulted in the successful generation of the �ERKO mice, which also survive to adulthood and exhibit phenotypes unique from those of the �ERKO (47). However, splicing variants of the disrupted genes that may encode for receptors with decreased functional activity have been detected in small amounts in each of the respective ERKO models (as discussed below) and therefore complicate the interpretation of the nonlethality of the gene targeting. Nonetheless, we now know that a loss of full function of any one of the two ERs is neither lethal nor detrimental to embryonic and fetal development in both mice and humans. Perhaps the survival of the ERKO mice, the aromatase-deficient humans, and the ER�-deficient male will prompt a renewed effort among clinicians to suspect and investigate the possibility of estrogen insufficiency or resistance in patients not responding to conventional therapies. The ERKO mice provide broad and multiple advantages to the research efforts toward understanding the function
366 COUSE AND KORACH Vol. 20, No. 3 and mechanism of estogen action. Much of what is known about estrogen action was inferred from in vivo studies involving castration or the administration of ER antagonists or inhibitors of estradiol synthesis. These findings have been complemented by the vast knowledge gained from in vitro cell culture studies, employing chimeric and mutant versions of the ER, varied cell types, multiple combinations of promoter-reporter gene constructs, as well as synthetic agonists and antagonists. However, there are distinct disadvantages to these experimental schemes. Studies using aromatase inhibitors and/or estrogen antagonists are complicated by several factors, including variability of the compound to block the action of the natural hormone or enzyme. The effectiveness of various antagonists is highly dependent upon the animal model, the tissue or cell of study, the bioavailability of the compound at different target tissues, and the class of antiestrogen used (8). This dilemma is further complicated by the discovery of the ER�, since no known ER-selective agonists or antagonists have been characterized at an in vivo level. The limitations of in vitro cell culture experimental approaches are obvious and mostly based on their finite application to the whole animal. Therefore, the ERKO models provide a unique tool to investigate the role of the ER in the context of the whole animal, and equally important, during the complete life span of the animal. At their most fundamental level, the ERKO mice address the role of the ER in the development and normal physiology of all organ systems, as well as in carcinogenesis, toxicity, and aging. Furthermore, unlike the “castrate” model in which several hormones are removed from the system, the ERKO mice retain the capability to synthesize the gonadal steroids, including the natural ER ligand, estradiol. Therefore, the biochemical functions of estradiol and the ER can be investigated in the presence of presumably intact pathways for the other gonadal hormones. The presence of estrogens in the absence of ER also provides for the possibility of discovering pathways of estrogen action that are independent of nuclear ER, or mediated via previously unknown forms of the receptor. Additionally, although the majority of in vitro studies indicate that ER� and ER� may have redundant functions, their differences in tissue distribution and response to certain ligands indicate the presence of distinct roles fulfilled by each. The fact that the �ERKO mice exhibit an unaltered pattern of ER� mRNA expression strengthens the usefulness of this model to dissect these potential ER�-mediated actions (93, 121). Finally, consistent with those criteria discussed earlier for establishing an endocrine function to an organ, the ERKO animals now provide a null background available for transgenic reintroduction of the ER of other species, mutated ERs, or targeted ER expression to a specific tissue or cell type. A detailed description of the targeting scheme employed to disrupt the mouse ER� gene can be found in the initial description of the �ERKO mice (46). As shown in Fig. 1, a 1.8-kb insert possessing the gene for neomycin (neo) resistance under the control of the phosphoglycerate kinase (PGK) promoter and including a PGK-polyadenylation signal was inserted into a NotI site in exon 2 of an ER� gene fragment subcloned from a genomic library of 129/J mouse DNA. The targeting insert was placed in a replacement � type targeting vector (122) with the appropriate ER� gene- flanking sequences. Upon successful targeting in mouse embryonic stem cells (129/J), the neo insert is placed approximately 270 bp downstream of the ER� translation start site and thereby inhibits proper expression of the ER� gene. Since this was the current state of the technology, no portion of the ER� gene was removed during the targeting event. Standard protocols of clone selection and blastocyst (C57BL/6J) injection were used to generate chimeric mice possessing the disrupted gene, some of which demonstrated germ-line transmission of the mutation when bred with wild-type mates (122). Southern blot and PCR analysis of genomic DNA from mice of all three genotypes indicated the correct targeting of the ER� gene and the absence of any heterologous recombination in other regions of the genome. Inbreeding of mice heterozygous for the ER� disruption resulted in a Mendelian distribution of all three genotypes as well as a balanced sex ratio, indicating that the ER� is not critical to sex determination at the level of the external genitalia (46). The generation of mice homozygous for a disruption of the ER� gene was similar to that described above for the �ERKO and can be found in detail in the initial description (47). A genomic clone that spanned a 15-kb region possessing the first three exons of the mouse ER� gene was selected from a 129/SvJ mouse library. A replacement � type targeting construct was generated to include 5� and 3� homologous sequences of 1.3 and 7.4 kb, respectively (Fig. 1). A PGK promoter-regulated neo gene was inserted in the reverse orientation into a PstI site in exon 3 of the ER� clone. Therefore, correct targeting resulted in disruption of the sequences coding for the first zinc finger of the ER� protein, a domain critical to normal function of the receptor. Chimeric and heterozygous offspring were generated as described above for the �ERKO mice. Once again, mice possessing the targeted disruption of the ER� gene were identified by diagnostic Southern blotting and PCR of genomic DNA. As with the �ERKO, inbreeding of mice heterozygous for the disruption yielded a Mendelian distribution of all three genotypes as well as a balanced sex ratio (47). In both knockout models, RT-PCR on RNA from target tissues indicated the presence of multiple splicing variants of the respective ER transcripts (47, 123). In neither case has wild-type-like mRNA transcribed from the disrupted receptor gene been detected. The greater proportion of the variants detected in each ERKO model possessed frame shifts that would result in a severely truncated or mutated ER if translated. However, in the �ERKO, a single-splice variant capable of encoding a mutant ER� protein with significantly decreased transactivational capacity in vitro was detected at very low levels (123). A similar ER� splice variant, in which the reading frame was preserved although coding sequences were removed, was detected in ovaries of the �ERKO mice. This single variant, if translated, would encode a mutant ER� lacking the first zinc finger and therefore would be unlikely to transactivate due to an inability to tightly associate with the chromatin structure within the regulatory regions of target genes. This is not the first report of targeted insertions resulting in aberrant splicing of a disrupted gene. <strong>Mice</strong> possessing a targeted disruption of the transforming growth factor-� gene produce a transcript in which the entire exon possessing the
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