Estrogen Receptor Null Mice - Endocrine Reviews

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June, 1999 ESTROGEN RECEPTOR NULL MICE 389 ER� mRNA in certain regions of the rat brain, including the medial nucleus of the amygdala and the periventricular preoptic nucleus. Therefore, with preliminary studies indicating distinct tissue localization of the two ERs in the reproductive tract, the brain may be the ideal tissue for the study of possible transactivational actions of ER�/ER� heterodimers. It is important to reiterate that studies have indicated a normal expression pattern for the ER� gene in the hypothalamus of the �ERKO mouse (93, 352). Aside from the receptor-mediated genomic actions of sex steroids that have been so well characterized, the possibility of nongenomic effects of gonadal hormones and their metabolites has also received increased attention. A number of rapid responses to gonadal steroids in various tissues have been reported and are believed to occur too soon after steroid exposure to be mediated by the classical mechanism of hormone nuclear receptors; therefore, they have been termed as being “nongenomic” (reviewed in Refs. 347, 355–358). These include the rapid activation of membrane calcium channels by progesterone in the maturing oocyte and spermatozoa, by estrogens in myometrial cells, and by androgens in rat osteoblast cells (347, 355, 356). Descriptions of similar nongenomic effects of steroids in the neuroendocrine system include rapid increases in cAMP levels in neurons, modulation of the GABA-GABA A receptor function, release of GnRH and dopmamine from nerve terminals, modulation of oxytocin receptors, and the release of PRL from GH3/B6 pituitary cells (343, 356, 357). Supportive experimental findings indicate the presence of membrane steroid receptors, including those for estradiol, in various cell types (359–361). Evidence that a membrane ER is structurally similar to the nuclear ER� was provided by Pappas et al. (362) in which multiple ER�-specific antibodies were shown to detect and localize ER� immunoreactivity in the cell membrane. Furthermore, Blaustein describes findings of extranuclear ER� immunoreactivity in the cytoplasm, dendritic processes, and axon terminals of neurons and suggests an active role for these receptors in neurotransmitter release (reviewed in Ref. 338). Recently, Razandi et al. (363) reported the detection of membrane ER� and ER� receptors in Chinese hamster ovary (CHO) cells transfected with an expression vector of the respective receptor cDNA, indicating that the membrane and nuclear forms of each ER originate from the same transcript and exhibit similar affinities for estradiol. These studies further demonstrated that the membrane-bound ERs were G protein linked and able to elicit a variety of signal transduction events, including the induction of cell proliferation (363). In contrast, Gu et al. (364) recently employed the �ERKO mouse to illustrate that the documented rapid action of estradiol on kainate-induced currents in the hippocampus occurs in the absence of a functional ER� gene, nor does ICI- 182,780 have an inhibitory effect, suggesting that ER� is not involved as well. Therefore, the putative membrane receptor involved in mediating the neuronal effect of estradiol in the hippocampus described by Gu et al. appears to be distinct from the intracellular nuclear form of the ER as well as that described by Razandi et al. (363). Regardless, the ultimate function of the nongenomic signaling pathways of the gonadal steroids in the proper organization and function of the mammalian brain remains unclear. Therefore, the ERKO mu- tant mice provide an excellent model to not only study the role of the nuclear receptors, but also further the investigations of steroid hormone actions that may be nuclear receptor independent. A comprehensive review of the neuroendocrine system is beyond the scope of this discussion and has been reviewed in detail elsewhere (337, 347). However, as was expected, distinct phenotypes in the neuroendocrine system have become evident in mice after disruption of the ER� gene. The ultimate consequences of the lack of ER� action in the neuroendocrine system are manifested in the ovary of the �ERKO female and as severe deficits in sexual and field behavior in both sexes of the �ERKO mice. Due to the relatively short time in which the �ERKO model has been available for study, no detailed characterizations of possible phenotypes in the hypothalamic-pituitary axis of this model have been carried out. Therefore, this section of the review will concentrate on what is currently known about the �ERKO, but will attempt to shed light on the possible distinct roles of both ERs based on the limited observations of the �ERKO. A. Hypothalamic-pituitary axis The hypothalamus may be thought of as the interface between the central nervous system and the endocrine system, i.e., the pituitary. The anatomical location of the hypothalamus, forming the base of the brain and residing just above the pituitary, is conducive to a function of translating neuronal signals from the brain into humoral factors that stimulate the appropriate actions in the anterior pituitary (365). The two components are connected by the hypothalamo-hypophyseal portal system, within which blood flows predominantly from the hypothalamus to the anterior pituitary, carrying the appropriate hormonal factors (365). These hormones act as releasing or inhibiting factors to control the secretory activity of the pituitary. In contrast, the posterior pituitary is connected directly to the hypothalamus via neurons passing through the pituitary stalk and functions as a storage organ for the hypothalamic hormones, oxytocin and vasopressin. The anterior pituitary is composed of at least five distinct cell types, all derived from a common primordium, which have been categorized by the particular peptide hormone they produce and secrete. These cell types are as follows, with the secretory hormone in parentheses: gonadotrophs (FSH and LH), corticotrophs (ACTH), thyrotrophs (TSH), somatotrophs (GH), and lactotrophs (PRL). Early studies employing steroid autoradiography demonstrated estrogen binding to varied degrees throughout the different cells of the anterior pituitary, although discrepancies among species are evident (reviewed in Ref. 339). These studies have been followed by those using in situ hybridization and immunohistochemistry, indicating that the majority of estradiol binding in the anterior pituitary is due to the expression of ER� (366, 367). In most species described, gonadotrophs and lactotrophs exhibit the greatest level of ER� followed by lower and varied levels of localization to the other cell types (339). Furthermore, Shupnik et al. (29, 368) have described in the rat pituitary a number of ER� mRNA isoforms characterized by
390 COUSE AND KORACH Vol. 20, No. 3 the removal of single or multiple exons as well as 5�-sequences that exhibit little homology to the full-length wildtype ER�. Although a distinct function for these ER� isoforms has not yet been determined, their transcription as well as that of the full-length wild-type ER� gene appears to be regulated by ovarian steroids (368, 369). Further complexity has been introduced by several recent descriptions of the presence of ER� mRNA in the anterior pituitary of the human (100), monkey (96), and rat (70, 98, 99). Wilson et al. (98) report that ER� mRNA levels were easily detectable in the pituitary of the 15-day-old rat, exceeding the levels of ER� mRNA. These levels decreased in the adult, whereupon a distinct sex difference became evident in which the anterior pituitary of the female expressed much higher levels of ER� mRNA (98). Still, previous studies employing radiolabeled ligand do not corroborate this difference in the rat anterior pituitary between the sexes at the level of estradiol binding (370, 371). Furthermore, there are contrasting reports concerning the cellular localization of the ER� transcripts in the rat anterior pituitary. Mitchner et al. (99) reported varied levels of ER� mRNA throughout the different cell types of the anterior pituitary. In contrast, Wilson et al. (98) describe the detection of both ER� and ER� mRNAs in the gonadotrophs, whereas the lactotrophs appear to possess ER� only. Nonetheless, as previously mentioned, we find low to undetectable levels of ER� mRNA in the pituitary of the adult mouse, including that of the �ERKO (93). However, in light of the above studies described in the prepubertal rat, which indicate a possible developmental role for the ER�, similar assays in the mouse are warranted. Although the rate of gonadotropin secretion from the anterior pituitary is directly modulated by the hypothalamus, the level of circulating sex steroids is the most important physiological determinant of serum gonadotropin levels in animals and humans (reviewed in Ref. 251). The positive and negative regulatory loops of the hypothalamic-pituitary-gonadal axis have been reviewed in detail and will be summarized briefly here (251, 362, 372). In short, the hypothalamus stimulates the synthesis and subsequent secretion of the gonadotropins into the circulatory system, which then act to induce gametogenesis and steroidogenesis in the gonads. The functional gonadotropins, FSH and LH, are composed of dimers of the common �-glycoprotein subunit (�GSU), with a distinct �-subunit that confers specificity to the hormone, i.e., active FSH consists of a FSH-� (FSH�)/�GSU dimer, whereas LH consists of a LH-� (LH�)/�GSU dimer (373). Hypothalamic stimulation of the anterior pituitary is via the release of GnRH, a deca-peptide that functions to positively regulate the synthesis and secretion of the gonadotropins from the anterior pituitary (365). A reciprocal hypothalamic factor that may act to inhibit pituitary secretion of gonadotropins has not yet been found. Hypothalamic secretion of GnRH is not tonic but rather in the form of pulses, driven by a poorly understood oscillating pulse generator (374, 375). The pulsatile stimulation of the anterior pituitary by GnRH thereby results in pulsatile gonadotropin release, which has been shown to be necessary for gonadal function and reproductive success (374, 375). Gonadotropin stimulation of the gonads subsequently results in gametogenesis and the synthesis of gonadal steroid and peptide hormones, which then feed back to the hypothalamus and pituitary to regulate FSH and LH secretion (251). However, differences in the system of feedback loops are apparent between the sexes. While there exists a tonic system to maintain a constant level of gonadotropins in both sexes, the female has been endowed with the ability to produce a surge of gonadotropin secretion to produce transient levels of hormone that are several fold higher than the tonic level (365). This massive gonadotropin surge provides for the reproductive cycle in the female and is critical to ovulation. 1. Female-negative gonadotropin regulation. There is ample experimental evidence in several species that estradiol can suppress the secretion of gonadotropins from the anterior pituitary (reviewed in Refs. 251, 372, 373, 376, and 377). Ovariectomy of the female rodent is known to result in significant elevations in serum FSH and LH that can be returned to intact levels with physiological treatments of estradiol (376, 378, 379). The effects of ovariectomy are mirrored in the gonadotrophs of the anterior pituitary, which exhibit equally elevated mRNA levels for the gonadotropin subunit genes (373, 376). Studies in the female rat indicate that by 21 days post ovariectomy, LH� mRNA levels rise to as high as 20fold, whereas the increases in FSH� and �GSU mRNA levels plateau at 4- to 5-fold (373). Daily treatments with estradiol will return the gonadotropin subunit mRNAs to the pregonadectomy levels within 7 days in the rat (373, 376). The ability of estradiol to maintain a tonic level of gonadotropins via regulation of both the expression of the gonadtropin subunit genes and the ultimate secretion of the peptide hormones is well accepted. However, the precise site at which the steroid exerts this effect has been difficult to ascertain. This is partly due to the fact that the ER has been detected in both the hypothalamic regions controlling pituitary function as well as the anterior pituitary itself, as previously discussed. Although there is evidence to support a direct effect of estradiol on both components of the neuroendocrine axis, it is believed that the hypothalamus may be the primary site of action in the negative feedback actions (reviewed in Refs. 251, 372, 373, and 376). In the laboratory animal, it is believed that castration principally results in an increased frequency of GnRH pulses and therefore increased tonic levels of gonadotropins, both of which can be restored to normal with exogenous estradiol replacement (373, 380– 383). Numerous studies have produced data to indicate that estrogen regulation of hypothalamic GnRH secretion may be the predominant pathway by which transcription of the gonadotropin subunit genes and gonadotropin secretion is maintained (373, 376). The most prominent include the characterization of transgenic mice that possess, within the anterior pituitary, a measurable reporter gene under the regulation of a gonadotropin subunit gene promoter, including the promotor of the human �GSU gene (384), the rat �GSU gene (385), and the bovine LH� gene (386). As expected, ovariectomy of the transgenic females resulted in increased transcription and activity of the transgene reporter gene. Keri et al. (386) illustrated that estradiol was able to reduce the postovariectomy rise in transcription of the reporter gene despite the lack of detectable DNA binding of the ER� to the
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Estrogen Receptor Null Mice - Endocrine Reviews

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