Estrogen Receptor Null Mice - Endocrine Reviews

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June, 1999 ESTROGEN RECEPTOR NULL MICE 397 The elevated levels of infanticide and aggressive behavior exhibited by the �ERKO females may be contributed to by the elevated levels of testosterone secreted by the acyclic ovary (Table 2). As discussed earlier, experimental evidence suggests that disruption of the ER� gene has resulted in a hypothalamic-pituitary axis with an enhanced capacity to respond to androgens in the �ERKO male. In support of this possibility, Ogawa et al. (418, 426) reported their preliminary finding of increased androgen receptor levels in the brain of the �ERKO female as early as 12 days of age. 2. �ERKO male. Given the apparent role of ER�-mediated estrogen actions in the masculinization of the brain, it was expected that the �ERKO males would exhibit a female-like behavioral phenotype. Surprisingly, however, Ogawa et al. (318) observed that a lack of hypothalamic ER� during development has little effect on the sexual behavior of the intact �ERKO male in terms of mounting and sexual attraction toward wild-type females. In contrast, Wersinger et al. (427) report that tests of male sexual behavior carried out in a neutral arena, as opposed to the male’s home cage as done in the study of Ogawa et al., demonstrates that the number of mounting attempts exhibited by the �ERKO males is reduced. Interestingly, the studies of Ogawa et al. illustrated that �ERKO males, however, exhibit an almost complete lack of intromission and ejaculation, even though the number and frequency of mounts were similar to those of wild-type males (318). Furthermore, treatment of castrated males with estradiol or the nonaromatizable androgen, DHT, resulted in no differences in sexual behavior compared with the findings in the intact �ERKO males (428). These results differ from those described by Ono et al. (429) and Olsen (131) in the androgeninsensitive Tfm mouse, which exhibits no male-like sexual behavior including a lack of mounting as well as intromission and ejaculation. However, the �ERKO and Tfm males are similar in terms of exhibiting complete insensitivity to the effects of both estradiol and testosterone as behavioral activators during adulthood. The �ERKO male behavioral phenotype described above is obviously a contributing factor to the infertility that results after disruption of the ER� gene. The culmination of the studies indicate that a discrete component of sexual behavior in the male mouse, i.e., consummatory activity, is dependent on the actions of ER�, whereas testosterone or possibly ER�mediated estradiol actions may regulate motivational aspects (428). Although reports of categorical studies on sexual behavior are not available, both the �ERKO (47) and ArKO (257) male mice appear to be fertile and able to sire multiple litters, suggesting a minor role for ER� in sexual behavior. The possibility of compensatory mechanisms mediated by ER� in the �ERKO cannot be ruled out. It is interesting, however, that the ArKO males, presumably lacking physiological levels of estradiol throughout life, show no obvious deficits in sexual behavior that result in infertility (257). It might be expected, given the apparent need of ER�-mediated estrogen actions illustrated by the �ERKO, that the ArKO male would display a similar phenotype. Perhaps, exposure to maternal steroid hormones during gestation in the ArKO mouse has allowed for the proper “organization” of the neuronal circuitry regulating sexual behavior. More detailed studies may elucidate subtle behavioral phenotypes that exist in both the �ERKO and ArKO models and will help further define the precise role that estradiol and testosterone play in the regulation of sexual behavior. A dichotomy similar to that observed for the elements of male sexual behavior in the �ERKO is observed when behavioral assays for aggression and parental instincts are considered. Intact �ERKO males demonstrate a relatively normal pattern of parental behavior as measured by levels of infanticide when placed in the presence of newborn pups (428). However, despite the fact that �ERKO males possess serum testosterone levels that exceed the norm by as much as 2-fold and show no reduction in the levels of AR (430) or ER� (93, 352) in the brain, they consistently exhibit a significant deficit in all male aggression indices tested (428, 430). Therefore, as in the case of certain components of sexual behavior, ER�-mediated actions appear critical to the development and/or activation of aggressive behaviors, whereas parental instincts appear to be independent of ER� action (428). VII. Phenotypes in Peripheral Tissues The ER and the estrogen-signaling pathway have been described in several peripheral organ systems (reviewed in Ref. 431). A discussion of the phenotypes that may occur in each of these after disruption of either of the ER genes is beyond the scope of this review. Therefore, we have chosen to focus on three areas, all of which have received great attention as sites of estrogen action that are critical to human health. These are the bone, cardiovascular system, and adipogenesis. Furthermore, studies toward specifically defining a role that ER may play in mediating the actions of estrogen in each of these systems have begun to employ the �ERKO, and eventually the �ERKO. A. Skeletal system The link between the onset of osteoporosis and the decreasing estrogen levels associated with menopause has been realized since the report of Albright et al. in 1941 (432). Postmenopausal estrogen replacement therapy is currently the most commonly prescribed drug treatment in the United States (433, 434). In most patients, the increased risks associated with long-term estrogen replacement therapy, such as breast and endometrial cancer, are strongly overshadowed by the well established reduction in the risk of osteoporosis and bone fracture (433). Bone is a dynamic tissue that is constantly being resorbed to serve as a mineral source for the body and remodeled to replace this reservoir as well as maintain skeletal strength. Osteoporosis is a defined pathology characterized by a loss in bone mass and strength and is believed to be due to a disruption in the equilibrium between bone resorption and formation (435). Current evidence supports the hypothesis that excess bone resorption occurs in the postmenopausal years, acting to strip the bone of mass and further remove the foundation upon which new bone may be formed (435). Several therapies are known to reduce the postmenopausal increases in bone resorption, including the intake of calcium and vitamin D, calcitonin,
398 COUSE AND KORACH Vol. 20, No. 3 bisphosphates, and estrogens (435). The obvious beneficial effects of estrogen replacement therapy have evoked an intense research effort for bone-specific estrogen agonists that lack the potentially harmful side effects in the breast and reproductive tissues. Ironically, the currently available “selective ER modulators” or SERMs, as these drugs have come to be termed, have been selected from the pool of nonsteroidal estrogen antagonists (reviewed in Refs. 8, 434, and 436). For several years it was believed that the effects of estrogens on bone physiology were indirect, inferred from the inability to detect ER within bone and bone cell cultures. However, in 1988, Komm et al. (437) and Eriksen et al. (438) simultaneously reported the detection of high-affinity, competable estradiol binding, ER� mRNA, and the induction of estrogen-responsive genes in cultured rat and human osteoblast-like cells, the bone-forming cell. We have reported similar findings, including the inhibition of estradiol transactivational activity with antiestrogens, in two separate osteoblast-like cell lines from the rat (439). Oursler et al. (440) have since demonstrated ER� and estrogenic activity in cultured avian osteoclasts, the bone-resorbing cell type, including estrogen induction of the genes for c-fos and c-jun. Using in situ RT-PCR analysis, Hoyland et al. (441) demonstrated the presence of ER� mRNA in both osteoblasts and osteoclasts in bone grafts from human females. Immunocytochemical methods have also been used to demonstrate the presence of ER� in multiple bone cell lines (442). Recently, Bodine et al. (443) reported significant increases in the levels of ER� transcripts during dexamethasone-induced differentiation of rat osteoblasts in vitro. Therefore, there is adequate experimental evidence to support the presence of a direct ER�-mediated estrogen-signaling pathway in bone. The discovery of the ER� introduced renewed vigor in the search for SERMs, allowing for the greater possibility of finding a receptor-selective agonist. The distinct expression pattern of the two ERs among various tissues has further enhanced the possibility of finding tissue-specific SERMs. Several recent studies have reported the detection of ER� in bone cells. Onoe et al. (444) employed RT-PCR to demonstrate the presence of both ER� and ER� mRNA in immortalized as well as primary osteoblast cell cultures from the rat. Similar to reports of ER�, both Onoe et al. (444) and Arts et al. (95) report significant increases in ER� mRNA levels during in vitro dexamethasone-induced differentiation of osteoblasts derived from the rat and human, respectively. Therefore, the generation of mice lacking ER� or ER� will once again prove invaluable in delineating the roles of the two receptors in bone physiology. The majority of animal studies concerning the role of estrogens in bone morphology and metabolism have been carried out in the rat (reviewed in Ref. 445). Ovariectomy in the rodent results in increased bone turnover similar to that seen in postmenopausal women; however, the mechanisms of action may differ between the species (445). The effects of ovariectomy in the rat include decreases in bone mineral density, cancellous bone area, and bone strength, whereas increases are observed in radial and longitudinal growth, osteoblast and osteoclast activity, and overall rates of bone turnover (445). <strong>Estrogen</strong> replacement, including those com- pounds with mixed agonist/antagonist activity, has been shown to reverse several of the effects induced by ovariectomy (445). However, the extent and direction of the changes induced by ovariectomy, as well as the protection provided by estrogen replacement, vary depending on the bone parameter, sex, and type of bone being evaluated, e.g., femur, tibia, calvaria, or vertebrae (445). Interestingly, a study of the androgen-resistant Tfm rat describes a bone phenotype similar to a wild-type female, i.e., shorter and thinner femurs, indicating that androgen action may also be critical to longitudinal and radial bone growth in the male rat (446). However, endogenous gonadal estrogens were able to maintain a normal cancellous bone mass in the Tfm rat (446). It is noteworthy that the first description of a human case of estrogen insensitivity due to a spontaneous mutation of the ER� gene exhibits severe osteoporosis as well as significant increases in longitudinal growth of bones (see Section VIII.C) (116). Unfortunately, few studies of the effects of steroids on bone physiology have been carried out in the mouse. Analysis of femoral bone length in �ERKO mice indicates a significant decrease in length and diameter in females and a slight decrease in males, when compared with age- and sexmatched wild-type controls (447). However, measurements of bone density and mineral content indicate the opposite effect, i.e., �ERKO males exhibited significant decreases throughout the femur (448), whereas the �ERKO females demonstrate just slight and localized decreases (447). In agreement with the ovariectomized rat model, �ERKO female mice exhibit increased bone resorption-remodeling rates (448). However, the decreased femur length observed in the �ERKO is in contrast to that reported in the ovariectomized rat and the ER�-deficient human male. Interestingly, a series of studies by Migliaccio et al. (449, 450) illustrated that prenatal and neonatal exposure to the synthetic estrogen, DES, also results in significantly shorter femur lengths as well as increased cortical bone thickness and increased trabecular bone at the epiphysis of the femur during adulthood in female mice. Therefore, it appears that in the mouse, aberrant estrogen exposure during development or a hereditary loss of ER� action leads to decreased longitudinal bone growth, contrasting experimental schemes resulting in a similar phenotype. These data indicate that pathways other than ER� may mediate the negative regulatory effects of estradiol on bone growth, suggesting a possible role for ER�. Longitudinal bone growth is a poorly understood process that depends on chondrocyte activity, including proliferation, hypertrophy, and the secretion of extracellular matrix at the growth plate (445). <strong>Estrogen</strong> is thought to slow this process by reducing the recruitment, proliferation, and synthetic activity of chondrocytes, thereby resulting in a maturation of the epiphyseal plate and inhibition of further longitudinal growth (445). The detection of both ER� (451) and ER� (452) in human epiphyseal chondrocytes has recently been described. Therefore, it is possible that ER�, in the context of significantly elevated levels of estradiol, results in an inhibition of long bone growth in the �ERKO mouse. It is also possible that the significantly elevated levels of serum androgens in the �ERKO female may be playing an influential role. This may
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Estrogen Receptor Null Mice - Endocrine Reviews

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