The estrogen receptor (ER) is thought to play a crucial role in the regulation of many life processes, including development, reproduction and normal physiology. Because there have been no known mutations of the estrogen...The estrogen receptor (ER) is thought to play a crucial role in the regulation of many life processes, including development, reproduction and normal physiology. Because there have been no known mutations of the estrogen receptor in normal tissue of humans and animals, its presence and tissue distribution is thought to be essential for survival. Using the techniques of homologous recombination, we have disrupted the ER gene and have produced a line of transgenic mice possessing the altered ER gene (ERKO). The mouse ER gene was disrupted by inserting a 1.8 kb PGK-Neomycin sequence into exon 2, approximately 280 bp downstream of the transcription start codon. The correct targeting of the disruption was demonstrated by Southern blot analysis and PCR. Western blot analysis of uterine preparations from ERKO females showed no detectable ER protein. Heterozygotes had one half the level of ER protein compared to wild-type animals. Estrogen insensitivity was confirmed using estrogen agonists, estradiol, hydroxy tamoxifen, diethylstilbestrol treatment for 3 days which resulted in a 3-4-fold increase in uterine wet weight and vaginal cornification in wild-type females, while ERKO mice were totally unresponsive. These data were further supported by the failure of estrogen or EGF treatment to induce DNA synthesis in uterine tissue of similarly treated mice. Lactoferrin, an estrogen-responsive gene in the uterus, was also assayed by Northern blot. Wild-type mice treated with a single estradiol injection showed a 350-fold induction in lactoferrin mRNA. while ERKO females showed no detectable response. Both male and female animals survive to adulthood with normal gross external phenotypes. As expected, females are infertile and demonstrate hypoplastic uteri and hyperemic ovaries with no apparent corpora lutea. Males are also infertile, with atrophy of the testes and seminiferous tubule dysmorphogenesis. Although the reproductive capabilities have been altered with a dramatic effect on the gonads, prenatal development of the reproductive tracts of both sexes appear to be independent of an ER-mediated response. Analysis of the mammary glands of the ERKO females at 4 months of age showed a primitive ductal rudiment rather than the fully developed ductal tree seen in wild-type siblings. Also absent were the terminal end buds seen during normal ductal morphogenesis. Both sexes show a decrease in skeletal bone density, supporting a direct role for ER action in bone. A single patient is described who is homozygous for a point mutation in the human ER gene at codon 157. The mutation produces a truncation of the ER protein and results in estrogen insensitivity syndrome. Most significant of the clinical findings are effects on skeletal bone density and retarded bone age. Findings from the patient and mice suggest that the absence of functional ER is not lethal. Mutation in the ER gene is present in the human population. Further characterization of the mice and identification of additional patients will be required to more fully understand the consequences of ER gene mutations.
Matzuk MM, Kumar TR, Shou W
… +4 more, Coerver KA, Lau AL, Behringer RR, Finegold MJ
Recent Prog Horm Res
· 1996 · PMID 8701077
With the advent of gene targeting in pluripotent mouse embryonic stem cells, it is now possible to modify the mammalian genome to generate mutant strains of mice with precise genetic mutations. The major goal of my labor...With the advent of gene targeting in pluripotent mouse embryonic stem cells, it is now possible to modify the mammalian genome to generate mutant strains of mice with precise genetic mutations. The major goal of my laboratory is to generate transgenic mice to use as physiologic models to study mammalian reproduction and development. The initial focus of our research has been to generate mice deficient in inhibins, activins, activin binding proteins (i.e., follistatin), and activin receptors (i.e., activin receptor type II) to understand their interactions and roles in the hypothalamic-pituitary-gonadal axis and mammalian development. Inhibins and activins, dimeric members of the TGF-beta superfamily, were discovered due to their role in pituitary follicle stimulating hormone homeostasis. However, these proteins have later been shown to have diverse endocrine, paracrine, and autocrine functions. Activins have been shown to mediate their signals through type I and type II serine/threonine kinase receptors. The high interspecies conservation of activins, inhibins, and activin receptors and the universal presence of activins in mammals, birds, amphibians, and fish suggest an evolutionarily conserved role of these proteins in animal development. Our initial studies have demonstrated a tumor suppressor role of inhibin in the gonads and adrenals and have also suggested a role of activins in cancer cachexia-like syndrome. To further study the gonadal tumor development and the cancer cachexia-like syndrome in these mice, we have begun to generate mice with multiple genetic alterations (e.g., mice deficient in both inhibin and Mullerian inhibiting substance). We have also generated mice deficient in other components of this complex system (e.g., activin beta A, activin receptor type II, follistatin). Analysis of these transgenic mutant models has aided our overall understanding of the critical roles these proteins play in the development of the reproductive system, in the modulation of the endocrine milieu that regulates reproductive function, and in mammalian development.
Thyroid hormone (T3) fulfills diverse functions in vertebrate development and physiology. These functions are thought to be mediated by two genes encoding the related T3 receptors. TR alpha and TR beta. The use of homolo...Thyroid hormone (T3) fulfills diverse functions in vertebrate development and physiology. These functions are thought to be mediated by two genes encoding the related T3 receptors. TR alpha and TR beta. The use of homologous recombination in embryonic stem cells to generate defined, single-gene mutations provides a powerful means to investigate the individual functions of TR alpha and TR beta in mice. We have shown that targeted inactivation of the TR beta gene results in goiter and elevated levels of thyroid hormone. Thyroid stimulating hormone (TSH), which is released by pituitary thyrotropes and is normally suppressed by increased levels of thyroid hormone, was present at elevated levels in homozygous mutant (Thrb-/-) mice. These findings suggest a unique role for TR beta that cannot be substituted by TR alpha in the T3-dependent feedback regulation of TSH transcription. Thrb-/- mice provide a recessive model for the human syndrome of resistance to thyroid hormone (RTH). Typically, RTH is associated with dominant mutations in TR beta. It is unknown whether TR alpha, TR beta, or other receptors are targets for inhibition in dominant RTH; however, the analysis of Thrb-/- mice suggests that antagonism of TR beta-mediated pathways underlies the disorder of the pituitary-thyroid axis. Thrb-/- mice also display defective maturation of auditory function, demonstrating that TR beta is essential for the development of hearing. Interestingly, hearing defects are generally absent in dominant RTH, indicating that in the auditory system, a dominant TR beta mutant cannot mimic the defect caused by loss of TR beta. This suggests the existence of tissue-specific mechanisms that modulate the activity of TR beta. These results define in vivo functions for TR beta and indicate that specificity in T3 signaling is conferred by distinct receptor genes.