Estrogen Receptor Null Mice - Endocrine Reviews

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June, 1999 ESTROGEN RECEPTOR NULL MICE 391 gonadotropin promoter sequences of the construct. Furthermore, the postovariectomy rise in promoter activity could be prevented by administration of a GnRH antagonist to the transgenic animal, thereby inhibiting GnRH action at the gonadotrope. Therefore, a loss of estradiol action via ovariectomy appeared to result in increased GnRH release from the hypothalamus, which in turn stimulated increased transcription of the reporter constructs. However, the possibility of estradiol actions at the level of the gonadotrope that may directly effect gene transcription or alter GnRH responsiveness can not be ruled out. Regardless of the precise site at which estrogens negatively regulate gonadotropin expression and release, genetic disruption of the ER� gene was expected to have effects in the female hypothalamic-pituitary axis that mimic ovariectomy. Northern blot analysis of RNA from pituitaries of intact wild-type and �ERKO females indicates this to be true. Scully et al. (282) demonstrated that in the �ERKO female, the levels of the �GSU transcript are elevated 4-fold, whereas FSH� and LH� mRNAs are as high as 7-fold compared with wild-type. Ovariectomy in wild-type female littermates produced elevated gonadotropin subunit mRNAs that approximated the levels observed in the intact �ERKO, indicating that the effects of an acute loss of estrogen action are similar to those produced by a hereditary loss of ER� (282). Therefore, despite the fact that the hypothalamic-pituitary axis of the �ERKO female is chronically exposed to elevated levels of estradiol, the significantly increased level of all three gonadotropin subunit transcripts resemble those of an ovariectomized female. These data provide strong support for a critical role of ER�, rather than ER�, in the negative regulation of transcription of the gonadotropin subunit genes. However, at this time, studies of the �ERKO have not provided data to further elucidate the precise mechanism or site at which a loss in ER� action has resulted in this effect. Although the transcript levels of both the LH�, FSH�, and �GSU are significantly elevated in the �ERKO pituitary, this effect does not extend to the serum levels of the gonadotropins. Whereas serum LH is elevated 4- to 7-fold in the adult �ERKO female, levels of FSH appear to be within the normal range (Table 2) (252). A similar effect is reported in the PRKO female mice, although the serum LH levels in this mutant female are not nearly as elevated as those in the �ERKO female (387). In addition, ovariectomy in the PRKO female results in a further elevation of serum LH (387), most likely due to the loss of serum estrogens. This effect is not observed in the �ERKO female (252), indicating that estradiol is the predominant steroid hormone maintaining tonic levels of LH in the female. As shown in Table 2, the serum gonadotropin levels in the �ERKO female indicate that only LH is significantly elevated, whereas FSH is within the wild-type range. This is in contrast to the levels of FSH� mRNA in the anterior pituitary of the �ERKO, which are elevated 7-fold and equal to those exhibited by an ovariectomized wild-type female. Furthermore, assays of pituitary homogenates from intact �ERKO females for FSH protein indicate levels within the wild-type range, suggesting that the divergence between gene expression and serum levels for FSH does not appear to be due to a decreased secretory rate of the hormone but rather at the level of translation. This is in contrast to the ArKO female mouse, in which serum levels of both gonadotropins are reportedly elevated 3-fold compared with the wild-type (257). There are a number of possible explanations for this observation in the �ERKO female. Whereas estradiol treatment of ovariectomized rats has been reported to completely block the expected increases in LH� mRNA and LH secretion, it appears to be only partially effective in reducing transcription of the FSH� gene and secretion of FSH (376). It is now known that FSH� gene expression and FSH secretion is selectively regulated in a positive or negative nature by the peptides activin and inhibin, respectively (reviewed in Ref. 388). This is illustrated in the knockout mouse model of the activin receptor type II gene, which exhibits suppressed FSH levels in the adult of both sexes, supporting a role for activin in the positive regulation of FSH secretion (248). Although it is believed that the reciprocal effects of the activin/inhibin peptides are mediated at the level of the anterior pituitary, the precise mechanisms of action remain unclear. Inhibin has been shown to alter the levels of GnRH receptor (389) and decrease FSH� mRNA levels (390–392), as well as inhibit translation of FSH� mRNA (391, 393). Activin appears to utilize similar mechanisms to exert opposite effects on FSH� mRNA transcription and translation and ultimate secretion of the hormone (394, 395). Therefore, the normal levels of FSH in the pituitary and serum of female �ERKO mice may indicate that disruption of the ER� gene has no effect on the pattern of activin and inhibin secretion. This is supported by the apparent negative regulation of FSH� at the translational level in the �ERKO female, suggesting the presence of an active inhibin-signaling pathway. Further support is provided by studies indicating that ovariectomy of the �ERKO female, and therefore a loss of ovarian inhibin secretion, results in elevated levels of serum FSH similar to those seen in ovariectomized wild types (252). Estradiol replacement was partially effective in reducing the serum FSH in the ovariectomized wild-type but completely ineffective in the ovariectomized �ERKO (252). These studies provide for at least two conclusions: 1) the partial effectiveness of estradiol in reducing serum FSH levels in the wild-type was likely via functional ER� in the hypothalamus that may complement the actions of inhibin in the intact female; and 2) ovarian factors other than estradiol, most likely inhibin, are maintaining normal serum FSH levels in the �ERKO female, possibly by mechanisms that override the loss of ER� action. It is also possible that both activin and inhibin synthesis and secretion may be altered in the �ERKO female, but do not result in a net difference in serum FSH levels. Future investigations to determine the serum levels of the activin/inhibin subunit peptides and their functional dimers in the �ERKO female are warranted. Another possible explanation for the selective increase in serum LH in the �ERKO female may be that a lack of ER� action has resulted in aberrations in the GnRH pulse frequency and amplitude that are more conducive to LH secretion from the anterior pituitary. Normal levels of �GSU transcripts can be maintained with constant GnRH stimulation of the anterior pituitary (376). However, normal expression of both gonadotropin �-subunit genes and gonadotro-
392 COUSE AND KORACH Vol. 20, No. 3 pin secretion requires pulsatile stimulation from the hypothalamus, and each responds differently depending on the amplitude and frequency of GnRH stimulation (373, 376). Varied expression of the GnRH receptor on the cell surface of the gonadotrophs also varies with the level of hypothalamic stimulation (396). This mechanism, by which the level GnRH receptors can be differentially regulated and thereby modify the gonadotropin responsiveness to GnRH, has been proposed to be a key element in the differential regulation of the two different gonadotropins by the same releasing hormone (397). Studies in the rat have revealed that more rapid GnRH pulses (15–60 min) favor the secretion of LH, whereas slower pulses (120 min) allow for secretion of FSH (251, 376). Positive regulation of LH synthesis and secretion is also more sensitive to the amplitude of GnRH stimulation (251, 376). Therefore, a loss of ER� action during development and maturation of the hypothalamic-pituitary axis may have resulted in a pattern of GnRH secretion that favors the translation and secretion of LH rather than FSH. The influence that ER� may have, including a possible compensatory role in the �ERKO hypothalamus, remains to be evaluated. 2. Female-positive gonadotropin regulation. In addition to maintaining tonic levels of serum gonadotropins, estradiol also plays a central role in the preovulatory gonadotropin surge mode in the female (reviewed in Refs. 263, 264, and 377). Differentiation of the neuroendocrine system results in the development of mechanisms necessary to produce a dramatic preovulatory rise in serum gonadotropins in response to the positive feedback of ovarian steroids. In the rodent, developmental differentiation of this pathway in the neuroendocrine system is unique to the female (365). The surge in serum LH and FSH is the hallmark of the female cycle and is critical to ovulation as well as the synchronized induction of appropriate sexual behavior and, therefore, is vital to the female ovarian cycle and fertility. Numerous studies have demonstrated that estradiol is required for the preovulatory gonadotropin surge, and that the timing and dose of estradiol exposure may be the most critical parameters (reviewed in Ref. 377). As with the negative regulatory effects of estradiol, the precise site of action for the sex steroids during the preovulatory gonadotropin surge also remains unclear. However, it is believed to be the result of the combined effects of external and internal cues transduced from the brain and the positive feedback of gonadal hormones that provide for a synchronized pattern of GnRH secretion upon an anterior pituitary that has been rendered transiently hypersensitive to the releasing hormone (reviewed in Ref. 377). In the monkey, destruction of the neurons involved in GnRH production can be overcome with pulsatile administration of exogenous GnRH, whereupon a gonadotropin surge can be produced with exogenous estradiol, indicating the pituitary as the predominant factor in the surge (398). However, this is not possible in the rodent, apparently due to a greater level of interdependency among the components of the neuroendocrine axis (365). Therefore, although the rises in ovarian estrogen secretion are critical to the generation of a gonadotropin surge, the pathways involved are poorly understood. A number of mechanisms involving direct actions of estrogen in the brain have been proposed and recently reviewed (264). However, it is unlikely that the actions of estrogen are via direct interaction with GnRH neurons since these processes appear to be devoid of ER (264). Therefore, the actions of estradiol appear to result in indirect stimulation of the hypothalamic neurons that synthesize and release GnRH. Possible mechanisms include steroid interaction with receptor-positive monoaminergic and opoid neurons that may mediate the ultimate effects to GnRH neurons, possibly via modifications in the levels of catacholamines, glutamate, �-aminobutyric acid, neuropeptide Y, �-endorphins, and galanin (reviewed in Refs. 263 and 264). At the level of the anterior pituitary, the preovulatory increases in estradiol may act in concert with GnRH to enhance gonadotrope sensitivity to the forthcoming rise in releasing hormone by increasing the levels of GnRH receptor (263, 377). Furthermore, the 5�-flanking region of the rat LH� gene has been shown to possess an imperfect estrogen-responsive element that is able to bind ER� and confer estrogen responsiveness to a chimeric promoter-reporter gene construct in vitro (399). Therefore, the estradiol-ER� complex may also act to directly increase LH� mRNA levels before the LH surge (251). Attempts to elicit an LH surge in the ovariectomized model with acute estradiol treatments have been reported to be only partially effective, indicating that ovarian factors other than estradiol are also required for a full physiological response (reviewed in Ref. 263). It is now known that the actions of progesterone and the PR are also a necessary component in the induction of the gonadotropin surge (reviewed in Ref. 263). The role of progesterone and PR in facilitating the preovulatory surge may be to induce a rapid release of GnRH from the hypothalamus, as well as possibly mediate a decrease in ER levels in the anterior pituitary, thereby possibly counteracting the inhibitory effects of estradiol (263). Although the precise mechanism of action may be unclear, the complete lack of a preovulatory surge in intact PRKO female mice provides strong support for the requirement of this steroid receptor (387). A cooperative role between the estrogen- and progestronesignaling pathways in the induction of the preovulatory surge may include the ability of estradiol to stimulate increased PR levels in both the hypothalamus and anterior pituitary, thereby increasing the sensitivity of these tissues to progesterone (400, 401). Shughrue et al. have shown that estradiol-induced increases in PR expression in the preoptic nucleus are possible in the �ERKO female (Fig. 8) and suggest that this may be a compensatory action of ER� (400). These same studies demonstrated that the preoptic nucleus of the hypothalamus in intact �ERKO females possessed a significantly greater level of PR mRNA when compared with wild-types, perhaps due to chronic stimulation of ER� by the elevated serum estradiol (400). Evidence to support this hypothesis is the apparent decrease in the level of PR transcripts observed after ovariectomy in the �ERKO, which can be returned to intact levels 6 h after a single treatment with estradiol (Fig. 8) (400). The preoptic area of the hypothalamus, more specifically, the anteroventral periventricular nucleus of the preoptic region (AVPV), is thought to play a critical role in transducing the gonadotropin surge via interactions with the GnRH neu-
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Estrogen Receptor Null Mice - Endocrine Reviews

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