Estrogen Receptor Null Mice - Endocrine Reviews

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June, 1999 ESTROGEN RECEPTOR NULL MICE 393 FIG. 8.In situ hybridization for progesterone receptor (PR) mRNA in female WT and �ERKO hypothalamus. A, PR mRNA was detected in the medial preoptic nucleus of wild-type (a) and �ERKO females (b) 5 days afer ovariectomy. Also shown is the increased detection of PR mRNA in ovariectomized wild-type (c) and �ERKO (d) females 6 h after treatment with 5 �g of estradiol. Asterisks indicate the third ventricle. B, Quantitative analysis of the hybridization signal shown in panel A. Note the dramatic increase in PR hybridization signal when ovariectomized (OVX) wild-type mice were treated with estradiol (E 2). Similarly, the hybridization signal seen in intact �ERKO females is attenuated by ovariectomy, but augmented to intact levels when ovariectomized females were treated with estradiol. Statistical significance is indicated as follows: **, P � 0.01, ***, P � 0.001. [Reproduced with permission from P. J. Shughrue et al.: Proc Natl Acad Sci USA 94:11008–11012, 1997 (400). © National Academy of Sciences, USA] rons. Unlike most sexually dimorphic nuclei, the AVPV is actually larger and composed of a greater number of dopaminergic neurons in the female compared with the male (402). In male rodents, this region is rendered inoperative by the actions of testosterone during differentiation (365), an effect that can be reproduced in females with neonatal testosterone or estradiol exposure (403, 404). Therefore, masculinization of this portion of the brain involves the destruction of a large portion of these neurons and is believed to be due to local aromatization of testosterone to estradiol and subsequent activation of ER-mediated pathways (405). In support of this hypothesis, Simerly et al. (405) reported that the AVPV region of �ERKO males possess a population of dopaminergic neurons more characteristic of a wild-type female, confirming a critical role of ER� in this differentiation process. Furthermore, the numbers of dopaminergic neurons in the female �ERKO are only slightly reduced when compared with wild-type, indicating a morphologically normal AVPV region (405). Therefore, with the �ERKO female exhibiting an apparent preservation of estrogen-induced increases in hypothalamic PR and a wild-type-like female phenotype in the AVPV region, it is conceivable that the hypothalamic mechanisms required for induction of the preovulatory surge may be intact. 3. Males: gonadotropin regulation. Because of the more prominent role of testosterone in the male, certain issues specific to the male hypothalamic-pituitary axis are worthy of discussion. Of course, lower aromatase activity in the testis results in circulating levels of estradiol in the male that do not approach those observed in the intact cycling female. Therefore, it would be expected that distinct mechanisms of steroid feedback and regulation of gonadotropin synthesis and secretion from the hypothalamic-pituitary axis have evolved in males, presumably one likely to be more dependent on testosterone. This difference is thought to occur at the level of the hypothalamus since the anterior pituitary generally exhibits no sexual differentiation (365) and possesses receptors for all sex steroids (339). A critical role of testosterone and AR-mediated actions in the negative regulation of gonadotropin secretion in the male is illustrated by the elevated serum LH levels in Tfm mice (301) and in humans with androgen insensitivity syndromes (38). As in the female, transcription of the gonadotropin subunit genes is significantly elevated after castration in the male, although peak levels are reached much earlier (�7 days) (373). Furthermore, FSH� mRNA levels appear to return to precastration levels by 28 days, whereas the levels of LH� and �GSU transcripts remain elevated (373). Estradiol is equally effective as testosterone in reducing serum LH levels that result after castration in the male (reviewed in Ref. 373). These data, along with the documented presence of P450 arom activity (reviewed in Refs. 406 and 407) and wide distribution of ER in the hypothalamic-pituitary axis support a role for locally synthesized estradiol and ER action in male gonadotropin synthesis and/or secretion (88, 339). Furthermore, adult male ArKO mice exhibit elevated levels of serum LH despite possessing significantly high circulating testosterone (257). Therefore, the roles of estradiol and testosterone often appear overlapping as well as distinct, making obvious the complexity of the steroid-feedback mechanisms that exist in the male. Any specific role the ER� may play in the regulation of gonadotropin synthesis and secretion in the male would be expected to become apparent in the �ERKO. Adult �ERKO males exhibit levels of hypothalamic GnRH, pituitary FSH� mRNA, and serum FSH that are within the normal range when compared with wild-type littermates (Table 2) (317). The normal levels of FSH� mRNA in the pituitary of �ERKO
394 COUSE AND KORACH Vol. 20, No. 3 males are in stark contrast to the significantly elevated levels found in the female �ERKO mice (282). This contrast between the sexes may reflect differences in the inhibin/activin levels or may represent a definitive sexual differentiation in the transcriptional regulation of the FSH� gene in the mouse, indicating that androgens are the primary acting steroids in the male. However, although not as extreme as those found in the �ERKO female, pituitary LH� mRNA and serum LH levels are increased 2-fold in the adult �ERKO male (Table 2) (317). In a series of experiments, Lindzey et al. (317) demonstrated that castration results in the expected elevated levels of serum LH in the wild-type, and a further increase in the already elevated LH levels in �ERKO males. The rise in serum LH that occurs upon castration even in the �ERKO male suggests that either estradiol-ER� or androgen-mediated mechanisms are maintaining the lower LH levels in the intact animal. Once again however, the mouse pituitary (including the �ERKO) appears to possess very little if any ER� mRNA (93), although ER� is expressed normally in the hypothalamic regions in the �ERKO (93, 352). Estradiol treatment of castrated animals over a period of 3 weeks reduced the serum LH levels to normal in the wild-type males, whereas no effect was observed in the �ERKO, indicating a requirement for ER� in this process (317). Although treatments of similar castrated males with testosterone was completely effective in producing an inhibitory effect on LH release in the wild-types, it was only partially effective in the �ERKO (317). The authors therefore concluded that the ability of testosterone to fully restore normal levels of LH in the sera of castrate wild-type males but only partially in the �ERKO males suggests that local aromatization of testosterone to estradiol and subsequent activation of ER�-mediated pathways act to enhance the negative feedback effects of androgens in the male hypothalamic-pituitary axis (317). However, the inability of testosterone to completely suppress the serum LH in the �ERKO male may be related to the dosage used in these studies. Strong evidence of AR-dependent regulation of LH secretion in the male �ERKO is found in preliminary experiments in which treatment with an antiandrogen (flutamide) increased serum LH by 3- to 10-fold in wild-type and �ERKO males, respectively. This suggests that �ERKO males have come to rely entirely on AR-mediated actions to regulate LH secretion, whereas the ER� continues to play a role in the wild-type. In these same studies, Lindzey et al. (317) illustrated that prolonged treatment with DHT, the more potent and nonaromatizable androgen, resulted in no reduction in the castrate levels of serum LH in wild-type but was partially effective in the �ERKO male. However, the DHT was effective in restoring hypothalamic GnRH content levels to normal in castrate males of both genotypes (317). Therefore, the enhanced effect of DHT in negatively affecting the hypothalamic-pituitary regulation of serum LH, including the inhibition of hypothalamic GnRH release, remains a puzzling phenomenom unique to the �ERKO male. It is possible that a lack of ER� action during development resulted in a “reorganization” of the hypothalamic-pituitary axis in the �ERKO male, and thereby somehow allowed for an increased sensitivity to androgens (317). Further studies in the �ERKO as well as the �ERKO males may help elucidate these unexpected results. 4. PRL regulation. PRL possesses more biological actions than all of the other anterior pituitary hormones combined. A recent review by Bole-Feysot et al. (279) thoroughly covered the current knowledge of the diverse actions of PRL, including its functions as a hormone, growth factor, neurotransmitter, and immunoregulator. A reflection of the multiple functions of PRL is the equally broad distribution of PRLbinding sites throughout the many physiological systems in vertebrates (279). The well known effects of PRL in reproduction include a critical role in the differentiation and function of the lactating mammary gland, as a luteotrophic hormone in the function of the corpus luteum and thereby as a promotor of blastocyst implantation, and an overall enhancement of the physiological functions in the tissues of the male reproductive tract (279). Nonreproductive roles of PRL include an involvement in osmoregulation; promotion of growth, development, and differentiation in several tissues; enhancement of metabolic activities in the brain, liver, pancreas, and adrenals; and various actions in immunoregulation (279). It has long been known that estradiol is a critical hormone in the regulation of PRL synthesis and secretion from the lactotrophs in the anterior pituitary. Estradiol has also been shown to stimulate lactotroph cell growth (reviewed in Ref. 281) and has been implicated as a possible factor in the promotion of PRL-secreting tumors in humans (reviewed in Ref. 339). Furthermore, the lactotrophs of several species have been shown to possess significant levels of ER�, strongly suggesting that the actions of estradiol are receptormediated (reviewed in Ref. 339). As discussed above, recent descriptions have indicated the presence of ER� in the lactotrophs of the rat anterior pituitary, although contrasting reports exist, possibly due to strain variations. Whereas Wilson et al. (98) describe the presence of only ER� in lactotrophs, Mitchner et al. (99) report variable levels of ER� mRNA throughout the various cell types of the rat anterior pituitary. Shupnik et al. (100) have also reported the detection of ER� transcripts in human PRL-secreting tumors. Once again, we find only low to undetectable levels of ER� mRNA in the pituitary of the adult mouse, including the �ERKO (93). The upstream regulatory sequences of the rat PRL gene have been found to possess an estrogen-responsive element that binds ER� and functions synergistically with the pituitary-specific factor, Pit-1, to promote expression (281, 408, 409). The required function of the ER� in the positive regulation of the PRL gene is nicely illustrated in the �ERKO mouse. The �ERKO females exhibit a 20-fold decrease in PRL mRNA levels in the anterior pituitary, whereas the �ERKO males exhibit a 10-fold decrease when each is compared with sex-matched wild-type controls (282). Although not as drastic, this reduction in the expression of the PRL gene is mirrored in the serum levels of the hormone in the �ERKO female, which possess an approximate 5-fold reduction in serum PRL (Table 2). Therefore, given the plethora of roles in which PRL is involved, it is likely that several of the phenotypes observed in the �ERKO mice may be due to a
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Estrogen Receptor Null Mice - Endocrine Reviews

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