Estrogen Receptor Null Mice - Endocrine Reviews

More documents

Recommendations

Info

June, 1999 ESTROGEN RECEPTOR NULL MICE 377 cells of antral follicles, were apparently preserved in the follicles of the �ERKO ovary. Follicular atresia in the ovary is a hormonally controlled process that is critical to oocyte selection. Although the factors that trigger atresia are not well understood, it is characterized by apoptosis of the granulosa cells of the follicle (reviewed in Refs. 223 and 224). Estradiol has been shown to be one of the many factors reported to protect the follicle from becoming atretic (250). Furthermore, androgens reportedly accelerate the process, and it may be an altered estrogen/androgen synthesis ratio in the follicle that leads to atresia (250). However, despite elevated androgen production, the presence of androgen receptor (AR) mRNA, and a lack of ER� action in the mature �ERKO ovary, an inordinate amount of apoptosis is not observed in the follicles (142). Estradiol has also been shown to facilitate the FSH induction of LH receptors in the granulosa cells of the mature ovulatory follicle in both in vivo (225, 231, 252) and in vitro experiments (229, 230). Nonetheless, the granulosa cells of the growing follicles, in addition to the enlarged cysts in the �ERKO ovary, possess significant levels of LH receptor mRNA when assayed by in situ hybridization (142). The most plausible explanation for these observations is that these estrogen actions are mediated by ER�, which has been shown to be expressed in a normal pattern in the granulosa cells of the �ERKO ovary, and concomitant with the expression of LH receptor (93, 142). Therefore, with data suggesting that disruption of the ER� gene did not result in an ovary completely refractory to estrogen, other aspects of ovarian physiology must be considered as possible factors in the etiology of the �ERKO ovarian phenotype. As previously discussed, ovarian function is tightly controlled by pituitary gonadotropins (see Section III.D.1). In turn, gonadotropin synthesis and secretion from the anterior pituitary are at least partially regulated by gonadal steroids acting via classical feedback mechanisms in the hypothalamic-pituitary axis (reviewed in Ref. 251). Indeed, disruption of the ER� gene has resulted in significant phenotypes in the hypothalamic-pituitary axis of the �ERKO female (see Section VI.A). Most notable is the increased and chronic secretion of LH in the �ERKO female that results in levels that are 4–7 times that found in wild-type females (Table 2) (252). As discussed above, synchronized increases in serum FSH and LH levels are critical to follicular maturation and ultimate ovulation in the ovary. However, it has been proposed that the follicular requirements for LH are finite, and the presence of abnormally high levels may force maturing follicles to either prematurely luteinize or become atretic (253). Therefore, the �ERKO ovarian phenotype is likely caused by the chronic exposure to abnormally high levels of LH. Support for this hypothesis can be drawn from a number of studies. Investigations involving prolonged treatment with antiestrogens over a period of at least 28 days have produced an ovarian phenotype in both mice (195, 196) and rats (197) that is similar to that of the �ERKO. Of course, interpretation of these studies is complicated by the ability of the antiestrogens to inhibit both ERs as well as estrogen action in both the ovary and hypothalamic-pituitary axis. However, these studies reported that the “�ERKO” phenotype of enlarged cystic follicles was produced only after prolonged treatments with those antiestrogens that possessed the ability to cross the blood-brain barrier and concurrently produce serum LH levels that were several fold higher than controls. Therefore, whereas the estrogen antagonists ZM-189,154 (197) and EM- 800 (195, 196) produced chronically elevated LH levels and an ovarian phenotype strikingly similar to that of the �ERKO, tamoxifen did neither (195–197). More definitively, Risma et al. (254, 255) report that targeted transgenic overexpression of the LH�-subunit in the mouse that results in a 15-fold increase in serum LH levels produces females that are anovulatory and exhibit an ovarian phenotype almost indistinguishable from that of the adult �ERKO female. Therefore, the similarity in the ovarian phenotypes described in the above studies, in which the models presumably possessed normal levels of ovarian ER�, combined with our observations in the �ERKO, strongly indicate that the ER� is not directly involved in the development of ovarian cysts due to hypergonadotropism. However, there are descriptions of at least two models in which serum LH is chronically elevated, yet do not manifest an ovarian phenotype similar to the �ERKO or those induced by antiestrogens or transgenics as described above. Female mice that are homozygous for a targeted disruption of the FSH�-subunit gene exhibit an approximate 5-fold increase in serum LH but do not show indications of enlarged cystic follicles in the ovary (241), indicating a role for FSH in this process as well. Bogovich (256) has provided supporting evidence by demonstrating that FSH is required along with prolonged exposure to LH (in the form of human CG) to induce follicular cysts in the rat. A likely role for FSH is the induction of LH receptor in the granulosa cells of the maturing follicles, thereby rendering the follicle responsive to the increased levels of LH. Another contrasting knockout model is that of the P450 arom gene (ArKO), in which the homozygous females possess significantly elevated serum LH and FSH levels but lack the capacity to synthesize estradiol (257). Although folliculogenesis is arrested at an antral stage in the ArKO ovary, no �ERKO-like cystic structures are reported (257). Therefore, assuming that a lack of estradiol synthesis would disrupt ligand-dependent activity of the ER� in the granulosa cells, the lack of ovarian cysts in the ArKO indicate an intraovarian role for estradiol in this phenotype. Therefore, although the �ERKO phenoytpe may be triggered by hyperstimulation of the follicles by LH, it is likely influenced by both FSH and ER� actions in the granulosa cells as well. Current studies utilizing prolonged treatment of �ERKO females with a GnRH antagonist to reduce serum gonadotropins are being carried out to further define the etiology of the ovarian phenotype (J. F. Couse, D. O. Bunch, J. Lindzey, D. W. Schomberg, and K. S. Korach, manuscript in preparation). Since the �ERKO ovarian phenotype develops and worsens only after sexual maturity, we have began studies to characterize ovarian function in the immature �ERKO female (J. F. Couse, D. O. Bunch, J. Lindzey, D. W. Schomberg, and K. S. Korach, manuscript in preparation). Although superovulation with exogenous gonadotropins was not successful in eliciting ovulation in the older �ERKO females, immature (�28 days) females do respond and produce viable oocytes that can be collected from the oviduct. However, the average number of oocytes collected from superovulated �ERKO females is significantly less than that yielded from
378 COUSE AND KORACH Vol. 20, No. 3 age-matched superovulated wild-type and heterozygous females. Therefore, intraovarian ER� action does not appear to be essential to ovulation when stimulated with exogenous gonadotropins. 4. �ERKO ovary. As discussed above, the �ERKO has provided a number of indications to suggest that intraovarian ER� action is not critical to ovarian function. Furthermore, several presumed functions of estradiol in ovarian granulosa cells appear to be preserved in the �ERKO, such as the attenuation of apoptosis and the induction of LH receptors. Based on the reported localization of ER� mRNA (49, 50, 63) and protein (69, 103, 104) to the granulosa cells of growing follicles, as well as the maintenance of a normal expression pattern for ER� in the �ERKO ovary (93, 142), it is likely that the newly discovered ER is the predominant mediator of estrogen action in the follicle. In the rat ovary, ER� is easily detectable by immunohistochemistry and exhibits an almost ubiquitous expression pattern within the granulosa cells of follicles ranging from the primary up to the ovulatory or Graafian stage (69, 103, 104). Byers et al. (63) demonstrated that ER� mRNA levels remain relatively constant during the follicular stage of the ovarian cycle but are rapidly decreased after the gonadotropin surge-induced luteinization of the granulosa cells. Interestingly, in 1975, Richards (216) reported a similar profile for high-affinity [ 3 H]-E 2 binding in the granulosa cells of the rat ovary, showing increased binding levels as follicles matured followed by marked decrease after luteinization. Because the affinities of the two ERs for estradiol do not significantly differ, it was not possible at that time to realize that the receptor being detected in the ovary was distinctly different (i.e., ER�) from that which was being concurrently described in the uterus (i.e., ER�). As stated previously, female mice homozygous for the targeted disruption of the ER� gene possess no gross aberrant phenotypes as neonates or during adulthood (47). However, during a continuous mating study of 8 weeks in which sexually mature wild-type or �ERKO females were housed with a known fertile wild-type male, a significant deficit in fertility in the �ERKO became obvious. As shown in Table 3, �ERKO females produced substantially fewer litters as well as significantly less numbers of pups per litter when compared with their wild-type littermates (47). Whereas the average litter size among wild-type females was 8.8 (�2.5) pups per litter, this number was reduced to 3.1 (�1.8) in �ERKO females (Table 3) (47). Furthermore, two of the tested �ERKO females yielded no litters, despite the observation of seminal plugs on multiple occasions, suggesting that abnormal sexual behavior was not a cause for the infertility. Gross TABLE 3. Fertility and superovulation data in the �ERKO female mice Genotype analysis of the uteri from the �ERKO females used in this study demonstrated no indication of embryo resorption during gestation. Therefore, the observed subfertility in the �ERKO females did not appear to be due to uterine dysfunction that results in premature termination of pregnancy. However, a possible defect in embryo implantation due to a lack of ER� could not be assessed in these studies. The nature of the subfertility in the �ERKO female described above strongly suggested an ovarian phenotype. Gross analysis of ovaries from �ERKO females indicated no distinct differences in size or morphology when compared with those of wild-type females. As shown in Fig. 5, histological analysis of ovaries from sexually mature �ERKO females illustrated the presence of a relatively normal interstitial compartment and follicles at various stages of the follicular cycle, ranging from primordial and primary to those with a clearly defined antrum, all possessing the expected thecal shell. Therefore, as demonstrated for ER� by the �ERKO, ER� does not appear to be essential for the establishment of germ cell number or ovarian development. Several of the speculated intraovarian roles of estrogen were discussed previously and include a proposed critical role in proliferation of the granulosa cells in the maturing follicle (217–220, 231). However, the multiple large follicles present in the �ERKO ovaries indicate no marked differences in granulosa cell number. Furthermore, serum estradiol levels in adult �ERKO females do not appear to differ from agematched wild-type females, at 24.2 (�3.3) and 30.5 (�2.8) pg/ ml, respectively. Biological evidence of estradiol synthesis in the �ERKO ovary is also illustrated by the apparently normal uterus and vagina, which exhibit the proper cyclic changes in morphology of a sexually mature wild-type mouse. Nonetheless, there were indications of an increased number of early atretic follicles and a sparse presence of corpora lutea in the �ERKO ovary when compared with the wild-type (47), suggesting that the observed subfertility in the �ERKO may be due to a reduction in completed folliculogenesis. A phenotype of compromised follicular maturation that more often terminates in atresia rather than ovulation, as described in the �ERKO female, is similar to that reported in a number of other mutant mice. As previously discussed, arrested folliculogenesis is described in knockout mice for other genes known to be expressed in granulosa cells, including the gene for FSH-receptor (242), P450 arom (ArKO) (257), IGF-I (245), and connexin-37 (247). It is therefore possible that a loss of ER� action has also resulted in alterations in the expression and/or function of one or more of these gene products. However, the normal serum estradiol levels and the appearance of estrogenic effects in the reproductive Continuous mating results Superovulation results n Litters per female (SEM) Pups per litter (SEM) n Oocytes per female (SEM) Wild-type 6 2.8 � 0.4 8.8 � 2.5 10 33.7 � 4.8 9–57 Heterozygous nd nd nd 11 52.5 � 5.7 a 20–77 �ERKO 11 1.7 � 1.0 a 3.1 � 1.8 b 11 6.0 � 1.5 a nd, Not determined. 0–13 a P � 0.05, Student’s two tailed t-test vs. wild-type b P � 0.001, Student’s two tailed t-test vs. wild-type Range
Page 1 and 2: 0163-769X/99/$03.00/0 Endocrine Rev
Page 3 and 4: 360 COUSE AND KORACH Vol. 20, No. 3
Page 19: 376 COUSE AND KORACH Vol. 20, No. 3

Estrogen Receptor Null Mice - Endocrine Reviews

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?