Estrogen Receptor Null Mice - Endocrine Reviews

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June, 1999 ESTROGEN RECEPTOR NULL MICE 403 istics associated with PCOS, also do not demonstrate this phenotype (254, 255). Several of the endocrine parameters associated with human PCOS are also observed in the �ERKO, especially the presence of elevated serum androgens and LH. Although the observation of hirsutism caused by elevated serum androgens is not possible in mice, analogous biological manifestations of elevated androgen levels are exhibited in the �ERKO female, including masculinized preputial (clitoral) glands and a thickened dermis. Furthermore, obesity is reported in a large proportion of PCOS patients and is thought to be a contributory factor in the extragonadal conversion of androgens to estrogens. As described above (Section VII.C), adult �ERKO females significantly outweigh their age-matched wild-type littermates because of excessive white adipose tissue. Interestingly, insulin resistance and hyperinsulinemia are often reported in PCOS patients and thought to contribute to further androgen synthesis in the ovarian thecum (204, 487). Taylor and Lubahn (490) reported an abnormal glucose tolerance test in �ERKO females that could be corrected with ovariectomy, suggesting PCOS-like insulin resistance. Therefore, the similarities of the endocrine abnormalities and ovarian phenotypes in the �ERKO and bLH�-CTP mice with those of human PCOS patients provide support that the cause may be extragonadal in nature, and possibly due to elevated LH in the presence of normal FSH. Although the initial cause of the abnormally high LH levels in �ERKO females may differ from that postulated in the PCOS patient, the resulting effects on the ovary are similar and may allow the �ERKO to be a useful experimental model in future investigations of this human disease. C. ER� and aromatase deficiency The first and only reported case of estrogen insensitivity in a human is that of Smith et al. (116) in which a male (46, XY) was determined to be homozygous for a single-point mutation in exon 2 of the ER� gene that resulted in a premature stop codon. Similar reports of human estrogen deficiency were also lacking in the clinical literature until recently. However, five cases of aromatase deficiency and therefore a lack of estrogen synthesis have been reported in both human males (119, 120) and females (117, 118, 120, 491). Therefore, the existence of humans that exhibit insufficient estrogen action due to either a lack of functional receptor or hormone, along with the successful generation of the ER null mice, suggests that estrogen is not essential to embryonic and fetal development in mammals. However, like the ER null mice, a distinct syndrome of phenotypes is apparent in both sexes of humans lacking in estradiol or ER. All three reports of females homozygous for inactivating mutations in the P450 arom gene describe pseudohermaphroditism, i.e., 46XX genotype, the presence of internal female reproductive structures but ambiguous external genitalia (117, 120, 491). This phenotype is due to the lack of aromatase activity in the fetus and placental unit during gestation, leading to the accumulation of androgens, which in turn elicit masculinizing effects on the fetus (117, 120, 491). A similar phenotype of ambiguous external genitalia was described in an infant in which placental aromatase deficiency was determined, although enzyme activity in the fetus was not evaluated at the time of the report (118). In the two cases of a complete lack of aromatase, the mother also exhibited virilization during pregnancy (120, 491), whereas a traceable level of placental aromatase activity appeared to inhibit this symptom in the case of Conte et al. (117). In all three cases, development of the internal structures of the female reproductive tract did not appear to be altered by a lack of estrogen action (117, 120, 491), although a compensatory role fulfilled by maternal estrogens cannot be ruled out. Serum levels of androgen, androgen precursors, FSH, and LH were elevated in the P450 arom-deficient patients as early as infancy (117, 491) and continued to be elevated as the females approached puberty (117, 120). At 12–14 yr of age, secondary development of the breasts, a pubertal growth spurt, and menarche were all absent, but virilization of the external genitalia was reported (117, 120). In all three cases, hyperstimulation of the ovary and the development of multifollicular cysts was illustrated, even as early as 2 yr of age (117, 120, 491). The ovarian pathology exhibited was similar to that observed in the �ERKO females and was compatible with a diagnosis of PCOS (120). Therefore, intraovarian estrogen action does not appear to play a role in the development of the multifollicular ovarian cysts in humans. <strong>Estrogen</strong> and progesterone replacement therapy alleviated the above phenotypes, resulting in normal gonadotropin and androgen levels, regression of the ovarian cysts, and in the two pubertal patients reported, the onset of breast development, a growth spurt, and menarche (117, 120, 491). The clinical syndromes exhibited by the two reported human male cases of aromatase deficiency (119, 120) and the single known human case of an inactivating mutation of the ER� gene (116) are strikingly similar. All three patients exhibited a normal onset of puberty and no gender-identity disorders, as well as normal external genitalia and prostate size (116, 119, 120). The patient lacking functional ER� exhibited a testicular volume and sperm density within the norm for men of his age (116). A normal testicular volume was also reported in the P450 arom-deficient patient of Morishima et al. (120). However, Carani et al. (119) reported small testis and severe oligozoospermia and infertility in the other male lacking aromatase, although the presence of similar findings in a brother with a normal P450 arom gene suggested other possible familial factors for this finding. Serum levels of androgens, FSH, and LH were all consistently elevated in the P450 arom-deficient males (119, 120), as well as estrone and estradiol in the ER�-deficient male (116). <strong>Estrogen</strong> replacement therapy resulted in the return of androgen and gonadotropin levels to the normal range in the P450 arom-deficient males. However, similar estrogen therapy induced no such changes in the ER�-deficient male (116). In fact, estrogen replacement therapy in the ER�-deficient male achieved circulating levels of the steroid that exceeded the norm by nearly 5-fold, yet no alleviation of the above pathologies or side effects were observed, such as impotence and gynecomastia (116). Alterations in the cardiovascular system have been reported recently in the ER�-deficient male, including dysfunctions in vasodilation (492) and premature coronary artery disease (493). The most overt phenotypes in all patients lacking sufficient estrogen action involved the skeleton. Females homozygous
404 COUSE AND KORACH Vol. 20, No. 3 for mutations of the P450 arom gene displayed a delay in bone age, a significant decrease in bone mineral density of the lumbar spine, and absence of the pubertal growth spurt (117, 491). Both the ER�-deficient male and the two males lacking aromatase were evaluated as adults and exhibited strikingly similar skeletal phenotypes. All three were characterized by tall stature (�95th percentile) with continued slow linear growth, a low upper/lower body segment ratio (�0.88, vs. 0.96, average for men), unfused epiphyses, bone age of 14–15 yr, and decreased bone density (116, 119, 120). Epiphyseal closure and increases in bone mineral density were observed after estrogen replacement therapy in the P450 arom-deficient patients (119, 120). However, as with the phenotypes described above in the ER�-deficient male, exogenous estrogen treatments resulted in no changes in bone physiology (116). Therefore, although it was once believed that estrogens were the major sex steroid influencing bone physiology in females and androgen fulfilled similar roles in the male, the phenotypes described above strongly indicate that estrogen action is critical to the pubertal growth spurt, bone mineralization, and epiphyseal maturation in both males and females. IX. Summary All scientific investigations begin with distinct objectives: first is the hypothesis upon which studies are undertaken to disprove, and second is the overall aim of obtaining further information, from which future and more precise hypotheses may be drawn. Studies focusing on the generation and use of gene-targeted animal models also apply these goals and may be loosely categorized into sequential phases that become apparent as the use of the model progresses. Initial studies of knockout models often focus on the plausibility of the model based on prior knowledge and whether the generation of an animal lacking the particular gene will prove lethal or not. Upon the successful generation of a knockout, confirmatory studies are undertaken to corroborate previously established hypotheses of the function of the disrupted gene product. As these studies continue, observations of unpredicted phenotypes or, more likely, the lack of a phenotype that was expected based on models put forth from past investigations are noted. Often the surprising phenotype is due to the loss of a gene product that is downstream from the functions of the disrupted gene, whereas the lack of an expected phenotype may be due to compensatory roles filled by alternate mechanisms. As the descriptive studies of the knockout continue, use of the model is often shifted to the role as a unique research reagent, to be used in studies that 1) were not previously possible in a wild-type model; 2) aimed at finding related proteins or pathways whose existence or functions were previously masked; or 3) the subsequent effects of the gene disruption on related physiological and biochemical systems. The �ERKO mice continue to satisfy the confirmatory role of a knockout quite well. As summarized in Table 4, the phenotypes observed in the �ERKO due to estrogen insensitivity have definitively illustrated several roles that were previously believed to be dependent on functional ER�, including 1) the proliferative and differentiative actions critical to the function of the adult female reproductive tract and mammary gland; 2) as an obligatory component in growth factor signaling in the uterus and mammary gland; 3) as the principal steroid involved in negative regulation of gonadotropin gene transcription and LH levels in the hypothalamic-pituitary axis; 4) as a positive regulator of PR expression in several tissues; 5) in the positive regulation of PRL synthesis and secretion from the pituitary; 6) as a promotional factor in oncogene-induced mammary neoplasia; and 7) as a crucial component in the differentiation and activation of several behaviors in both the female and male. The list of unpredictable phenotypes in the �ERKO must begin with the observation that generation of an animal lacking a functional ER� gene was successful and produced animals of both sexes that exhibit a life span comparable to wild-type. The successful generation of �ERKO mice suggests that this receptor is also not essential to survival and was most likely not a compensatory factor in the survival of the �ERKO. In support of this is our recent successful generation of double knockout, or ��ERKO mice of both sexes. The precise defects in certain components of male reproduction, including the production of abnormal sperm and the loss of intromission and ejaculatory responses that were observed in the �ERKO, were quite surprising. In turn, certain estrogen pathways in the �ERKO female appear intact or unaffected, such as the ability of the uterus to successfully exhibit a progesterone-induced decidualization response, and the possible maintenance of an LH surge system in the hypothalamus. Furthermore, it is apparent that several of the �ERKO phenotypes may be aggravated by the downstream attenuation of progesterone and PRL action or even enhanced by increased androgen sensitivity, including those observed in the mammary gland, gonads, bone, and behavior. In addition, disruption of the ER� gene may have unmasked estrogen-signaling systems that were not easily detectable in the wild-type, such as 1) the apparent estrogen actions of the 4-OH-E 2 in the �ERKO uterus that are independent of ER� and ER�; 2) the ability of estrogen to induce increased levels of hypothalamic PR in the �ERKO female; 3) estradiol-induced potentiation of select neurons of the hippocampus; and 4) the protective effects of estrogen in the carotid vascular injury model. Finally, the concurrent descriptions of human mutations resulting in a lack of estrogen action due to a loss in ligand or functional receptor have illustrated the utility of the ER null mice as a model system to further understand the pathologies that result. Therefore, the �ERKO mice have illustrated the several ways in which data collected from a knockout model can quickly contribute to the current knowledge concerning the function of a particular gene. However, it is worth noting the distinct differences in thought that occurred during the generation of the �ERKO and �ERKO models. At the time the work was initiated to generate the �ERKO mice, much was known about the many roles of estrogen and the ER; therefore, several educated predictions of the possible phenotypes were possible and have since been confirmed, rejected, or reevaluated. However, the conception of the �ERKO mice occurred only 2 yr after the discovery of the ER�. Because little was known about the function and role that the ER� might play, it was difficult to make sound predictions. There-
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Estrogen Receptor Null Mice - Endocrine Reviews

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