Estrogen Receptor Null Mice - Endocrine Reviews

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June, 1999 ESTROGEN RECEPTOR NULL MICE 395 direct loss of or simply enhanced by the concurrent decrease in PRL signaling. Interestingly, the extremely low levels of PRL mRNA in the anterior pituitary of the �ERKO female are even significantly less than that observed 14 days after ovariectomy in the wild-type (282). Therefore, the loss of ER� during development and differentiation of the lactotrophs in the anterior pituitary has resulted in a phenotype that is more severe than that induced by postpubertal ovariectomy, possibly due to a decrease in lactotroph cell number. It has been proposed that the lactotroph and somatotroph cell types of the adult anterior pituitary may be derived from a common cell that expresses both the genes for GH and PRL during development (reviewed in Ref. 410). The factors that may be involved in the terminal differentiation of this stem cell into a distinct cell type secreting only one of the respective hormones remain elusive. Because the appearance of the ER and the ontogeny of PRL expression appear to coincide in the developing pituitary, estrogen action has been proposed as a possible factor (410–412). However, a defect in the cell lineage of the lactotrophs that may be expected due to a loss of ER� action was not apparent in the �ERKO, as immunostaining for both PRL and GH localized expression of the genes to distinct cell types (282). Furthermore, estrogen has also been shown to stimulate proliferation of the lactotrophs and PRL-secreting cell lines (reviewed in Ref. 339). Therefore, since the marked difference in PRL mRNA levels observed between the �ERKO female and the ovariectomized wild-type is not apparently due to a defect in the differentiation of the lactotrophs, it may possibly be due to a decreased number of lactotrophs in the anterior pituitary of the �ERKO. Scully et al. (282) provided evidence against this hypothesis, by once again employing immunohistochemical staining to illustrate only a modest decrease in lactotroph cells in the anterior pituitary of the �ERKO mice. Therefore, ER� action does not appear to be required for either differentiation or proliferation of the lactotrophs in the mouse anterior pituitary. However, a recent report by Chun et al. (413) has illustrated a distinct contrast in the level of occupied ER required to elicit proliferation and that required for PRL synthesis in PR1 cells, a PRL-secreting cell line. Whereas approximately 50% of the cellular pool of ER was required to be complexed with estradiol for half-maximal stimulation of the PRL gene, only 0.1% was required to induce cellular proliferation (413). These results suggest that the mechanisms required for estrogen-induced lactotroph proliferation are hypersensitive in this cell line compared with the mechanisms involved in regulation of the PRL gene (413). Therefore, it is possible that the small amount of the active ER� splicing variant known to be present in the �ERKO (see Section II.C.) has allowed for sufficient estrogen signaling and lactotroph proliferation during develpment, resulting in the apparent lack of a somewhat expected phenotype of decreased lactotroph cell number in the pituitary of �ERKO mice. B. Behavior There are obvious effects of the gonadal steroids on sexual behavior in vertebrates; however, a more defined knowledge of these actions has become evident from a series of classical experimental schemes. These laboratory studies often relied on perinatal castration and/or developmental exposure to exogenous steroids followed by studies of the activational abilities of the different steroids during adulthood. The majority of such investigations have been carried out in the rat, but similar results have been described in other species (345, 414). Breifly, studies on sexual behavior in the rat have shown that 1) castration on the day of birth results in a feminized adult male that exhibits a female pattern of behavioral responses when treated with estradiol and progesterone, and 2) neonatal testosterone or estradiol treatment of a female results in a masculinized adult that exhibits a malelike pattern of behaviors and is refractory to estradiol and progesterone (131, 337). The culmination of the data collected from such experimental schemes has led to the conclusion that testosterone secreted from the perinatal testes during a critical developmental window results in permanent changes in the hypothalamic nuclei of the brain that mediate male sexual behavior. However, the data indicating that developmental exposure to estradiol results in an adult phenotype that is similar to that elicited by testosterone suggest that many of the masculinizing effects of perinatal testosterone may be via local aromatization of the hormone to estradiol and subsequent activation of the ER signaling pathway (reviewed in Refs. 406 and 407). In addition, estradiol is also necessary for normal development of the female brain, although in lower amounts (131). Therefore, sex steroid-mediated sexual differentiation of the various regions of the brain that are critical to behavior relies not only on the nature of the steroid ligand, but also on the dose and timing of exposure (348). Before the availability of the �ERKO mouse, McCarthy et al. (415) employed an elaborate technique of infusing anti- ER� oligodeoxynucleotides into the neonatal rat hypothalamus to elucidate a direct role for ER� in the sexual differentiation of the female brain. This experimental scheme was based on the hypothesis that the presence of specific ER� antisense oligodeoxynucleotides in the hypothalamus would interfere with proper expression of the ER� gene during a critical period of differentiation (415). The experimental groups included neonatal rats treated with testosterone plus or minus the infusion of the ER� antisense oligodeoxynucleotides. As adults, those females infused with the ER� antisense oligodeoxynucleotides exhibited more female sexual behavior compared with those treated with androgen alone. The investigators thereby concluded that the reduced ER� expression protected the infused female rats from the masculinizing effects of testosterone exposure (415), providing strong evidence that local aromatization and subsequent estradiol activation of the ER� pathway plays a primary role in the masculinization of the rat brain. However, the experimental scheme of McCarthy et al. does not allow for a direct comparison with the �ERKO female, due to the caveats discussed (see Section II.C.1). It is important to recognize that the �ERKO are deficient in ER� throughout development, whereas McCarthy’s scheme produced a lack of ER� action that was only transient and most likely not as complete. The use of technologies to target individual genes has created numerous models available for studies in the behav-
396 COUSE AND KORACH Vol. 20, No. 3 ioral sciences. A recent review by Nelson and Young (128) summarized and compared the behavioral or lack of behavioral phenotypes in a select 50 murine knockout models. Due to the lack of any grossly apparent behavioral phenotypes in the �ERKO mice, only those studies concerning the �ERKO will be discussed here. 1. �ERKO female. The dependence of female sexual behavior on the synchronized fluctuations in estradiol and progesterone that occur during the ovarian cycle have been described in detail (reviewed in Ref. 416). In the rodent, circulating estrogens continue to rise as ovulation approaches, eventually leading to the gonadotropin surge that not only triggers the release of the oocyte from the ovary but also a marked increase in serum progesterone. This dramatic rise in circulating progesterone is required for an optimal display of the lordosis posture, a measurable response required for successful copulation (416). The gonadotropin surge from the hypothalamic-pituitary axis is due to the postive-feedback actions of estradiol. The development of this pathway in the rodent is unique to the female as a result of sexual differentiation of the neurons in the anteroventral periventricular nucleus of the preoptic area that serve to regulate hypothalamic function (264, 365, 405). As discussed above, feminization of the brain involves the actions of estradiol during fetal and neonatal development that may rely heavily on the dose and time of exposure. Therefore, knockout models for ER� and ER�, as well as the PR, have and will continue to serve as invaluable resources for dissecting the role of each receptor-signaling system in female sexual behavior. In general, evaluation of the aberrant sexual behaviors of the �ERKO female must consider not only the absence of ER� signaling, but also the elevated levels of serum testosterone that exist in the adult female (Table 2). Despite the presence of the hormones presumably required for sexual behavior, intact adult �ERKO females exhibit behavior that resembles that of a male in terms of parental, aggressive, and sexual activities (417, 418). When placed in the presence of a stud male, �ERKO females display a complete lack of sexual receptivity, measured as prelordotic behavior and a lordosis posture (418). In fact, intact �ERKO females were often treated as intruders and attacked by the stud male (417). However, similar studies using ovariectomized females indicate that this behavior of the stud male was most likely elicited by the significantly elevated levels of circulating testosterone in the �ERKO female (418, 426). These same studies, employing ovariectomized females coupled with steroid replacement of varied combinations, illustrated a complete resistance to estradiol (418, 419) and a minimal effect of progesterone in inducing a lordosis response in the �ERKO female (418). Although the above studies have indicated a prominent role for ER� in sexual behavior in the mouse, the precise pathways disrupted by a lack of ER� remain unclear. Not surprisingly, the PRKO female mice also exhibit a lack of normal sexual behavior and are unable to produce a lordosis posture even when treated with doses of either estradiol and/or progesterone (44). However, this same study reported an inability of estradiol alone to induce lordosis but rather a requirement for both estradiol and progesterone in wild-type females (44). This is in contrast to the capacity of estradiol to solely induce lordosis in the wild-type controls employed by Rissman et al. (419) and may be a reflection of the differences in background strain and/or experimental design employed in the two studies. The �ERKO and PRKO models nicely illustrate a requirement for both estradiol and progesterone action for a full lordotic response. As early as 1939, estrogen exposure before progesterone treatment was found to be required for a full display of sexual behavior in the rat (420). As in the uterus, the expression and induction of PR in certain regions of the brain is under estrogen regulation. Studies have indicated that estradiol elicits detectable increases in PR in the hypothalamus, strongly suggesting that this estrogen-action is required for ultimate progesterone-induced sexual behaviors (346, 421, 422). Therefore, disruption of the ER� gene may be expected to cause abnormally low levels of PR expression in those areas of the brain that mediate female sexual behavior and therefore may explain the lack of such behavior in the �ERKO mouse. However, two separate reports have described the ability of estradiol to induce increases in PR transcripts in the forebrain of the �ERKO female mouse, including regions of the preoptic area (400) (Fig. 8) arcuate nucleus, caudal ventromedial hypothalamus, and posterodorsal medial hypothalamus (423). However, the extent of estrogen-induced increases in hypothalamic PR in the �ERKO female is slightly attenuated compared with that observed in similarly treated wild-type mice (400, 423). It is possible that this observed estrogen action in the �ERKO female is either mediated by a splicing variant of the disrupted ER� gene or by ER�. Regardless, the level of estrogeninduced PR in the �ERKO female does not appear to allow for a response to progesterone that is sufficient to elicit sexual behavior. These data support the conclusion that normal expression of female sex behavior requires the sequential activation of the ER�- and PR-signaling pathways. Significant deficits in parental behavior and a greater tendency toward infanticide is also observed in �ERKO females compared with wild-type littermates (418). These phenotypes do not dramatically differ between intact and ovariectomized �ERKO females; however, levels of infanticide were reduced in tests carried out after a prolonged postgonadectomy period (65 days) (418). The �ERKO female exhibits aggressive behaviors that are significantly elevated compared with the wild-type female littermates, and in stark contrast to the dramatically reduced aggressive behavior displayed by the �ERKO male (to be discussed below) (418). Remarkably, acute treatment of ovariectomized females with estradiol resulted in the expected reduction in aggressive behavior in both the wild-type and �ERKO females (418). Previous studies have shown that whereas both testosterone and estradiol can elicit aggressive behaviors in the castrated male mouse, only testosterone is effective in the ovariectomized female mouse (424, 425). These studies, in combination with the findings in the �ERKO, indicate that differentiation as well as activation of aggressive behaviors in the female mouse are testosterone dependent. However, the preserved ability of estradiol to reduce aggression in the ovariectomized �ERKO female is puzzling and may indicate an ER�-mediated pathway.
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