Estrogen Receptor Null Mice - Endocrine Reviews

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June, 1999 ESTROGEN RECEPTOR NULL MICE 375 roid being produced. The cell-specific and temporal actions of the gonadotropins, LH and FSH, regulate the type and activity of the steroidogenic enzymes expressed within the granulosa and the thecal cells. The model states that LH acting via the constituitively expressed LH receptor on the cell surface of thecal cells stimulates the synthesis of androgens (androstenedione) in the growing follicle. This requires the initial conversion of cholesterol stores to pregnenolone by the cholesterol side-chain cleavage enzyme (P450 scc) and is thought to be a rate-limiting step in thecal cell steroidogenesis (204). Still within the thecum, pregnenolone is converted to progesterone and then to androstenedione via the enzymatic actions of 3�-hydroxysteroid dehydrogenase and 17�hydroxylase/C 17–20 lyase (P450 17�), respectively (204, 206, 210). Regulation by LH has been shown to occur at both the transcriptional and translational levels for the P450 scc and P450 17� genes (206). Granulosa cells lack expression of the P450 17� enzymes required to produce androgens, the precursor of estradiol, and therefore are dependent on the passage of the thecal-derived androgens through the basement membrane and into the granulosa compartment. This cellular cooperation provides the basis of the two-cell portion of the model. The second gonadotropin, FSH, acts solely upon the granulosa cells to stimulate the enzymatic conversion of the androstenedione and testosterone to estrone and estradiol, via P450-aromatase (P450 arom), and 17�-hydroxysteroid dehydrogenase, respectively (204, 206, 210). The estradiol is then released into the follicular fluid, whereupon the bulk passes back through the basement membrane and enters the circulation. Upon ovulation, the luteal phase begins with luteinization of the follicle and differentiation of the remaining granulosa and thecal cells to form the corpus luteum. The relative amounts and activities of the steroidogenic enzymes are altered once again and shift toward synthesis of large amounts of progesterone. Recent data have challenged the “two-cell” model to incorporate the descriptions of a role for the oocyte in regulating granulosa cell steroidogenesis. Elegant in vitro experiments involving the surgical removal of the oocyte from isolated growing follicles have demonstrated the existence of an ooctye-secreted factor that is able to inhibit granulosa cell estradiol and progesterone synthesis (211–213). 2. Review of intraovarian estrogen actions. In 1940, both Pencharz (214) and Williams (215) independently reported a direct and specific ability of estradiol or DES to induce significant increases in ovarian weight in the hypophysectomized rat. These same seminal studies also described the synergistic effect of estradiol on gonadotropin-stimulated increases in ovarian weight (214, 215). Since then, numerous intraovarian effects of large amounts of locally synthesized estrogens have been described and postulated to be essential to normal follicular development and ovarian function. In granulosa cells of the growing follicle, estrogen has been reported to increase the levels of its own receptor (216), as well as induce DNA synthesis and proliferation (205, 217– 220), increase the number and size of intercellular gap junctions (221), stimulate synthesis of IGF-I (222), and attenuate apoptosis and follicular atresia (223, 224). Estradiol is also known to augment the actions of FSH on granulosa cells, resulting in the maintenance of FSH-receptor levels (218, 225–227) and the acquisition of LH-receptor (218, 228–231), an event critical to successful ovulation. Ultimately, the actions of estradiol act to enhance follicular responsiveness to gonadotropins and thereby result in increased aromatase activity and further estrogen synthesis (231, 232). Therefore, normal ovarian function appears to be dependent on a multitude of auto- and paracrine actions of estradiol that act in concert with the gonadotropins secreted from the anterior pituitary to provide for successful folliculogenesis and steroid production. Nonetheless, immunohistochemical detection and characterization of ER in the different ovarian compartments have proven difficult, although studies employing binding assays with radiolabeled ligands report the presence of ER in ovarian granulosa cells of the rat (216, 233–235), mouse (236), rabbit (236), and pig (235). The discovery of the ER� and its reportedly high mRNA levels in the ovary (49, 63, 93) reinforces the need for thorough immunohistochemical studies for the two distinct ERs in the ovary. Reports of localization of ER� and ER� transcripts in the rat ovary by in situ hybridization indicate the presence of low levels of ER� mRNA with no specific pattern (63), whereas ER� mRNA is easily detectable and predominantly localized to the granulosa cells of growing follicles (49, 63). Sar and Welsch (103) recently described immunohistochemical studies with ER�- and ER�-specific antibodies, reporting that ER� immunoreactivity is indeed highly expressed in and localized to the granulosa cells of growing follicles, whereas ER� staining appears limited to the interstitial/thecal cells in the rat ovary. Similar findings of immunohistochemical localization of the ER� to the ovarian granulosa cells were reported in the rat by Hiroi et al. (104) and in the cow by Rosenfeld et al. (69). Brandenberger et al. (94) reported the RT-PCR detection of ER� and ER� transcripts in normal and neoplastic human ovary and ovarian cell lines. This study further described the presence of easily detectable levels of ER� mRNA and very low levels of ER� mRNA in the granulosa cells, whereas the opposite was found in a cell-line derived from the ovarian outer surface epithelium (94). Misao et al. (237) also used RT-PCR to detect ER� and ER� transcripts in human corpus luteum. Both Misao et al. (237) and Byers et al. (63) demonstrated a downregulation of ER� mRNA during luteinization of the follicle and the differentiation of the corpus luteum in the human and rat, respectively. Interestingly, a report by Iwai et al., before the knowledge of ER�, described the detection of ER� immunoreactivity in the granulosa cells of the rabbit ovary (238), possibly illustrating another variation in the expression pattern of the two ERs among different species. Nonetheless, ER� and ER� are present in the adult rodent ovary. Therefore, disruption of the genes encoding these receptors may be expected to result in distinct ovarian phenotypes. In addition, the dissimilar expression patterns for ER� and ER� among the functional units of the follicle suggest that compensatory mechanisms fulfilled by the remaining functional gene in each respective ERKO may not be possible in the ovary. 3. �ERKO ovary. The ovary of the neonatal and prepubertal �ERKO female does not exhibit any gross differences when
376 COUSE AND KORACH Vol. 20, No. 3 compared with those of wild-type littermates (142). The mature �ERKO ovary possesses a normal complement of primordial follicles, indicating no defects in germ cell generation or migration to the gonad during fetal development. However, at the commencement of sexual maturity, it becomes apparent that the �ERKO female is anovulatory and exhibits a distinct ovarian phenotype of enlarged, hemorrhagic, and cystic follicles as shown in Fig. 4. These cystic structures often accumulate in the ovary, making the gonad appear grossly as a bundle of dark grapes, a signature phenotype of the �ERKO female. However, growing follicles in the tertiary and pre- to small antral stage are present in the mature �ERKO ovary, indicating that ER� is not required for the recruitment of primordial follicles and the initial stages of the follicular phase. This is in contrast to the described phenotypes of mice possessing a mutation of the steel (Sl/Sl t ) locus (239), or a targeted disruption of the genes encoding growth differentiation factor-9 (240), or the vitamin D receptor (45), all of which exhibit phenotypes of follicular arrest at very early stages. The growing and cystic follicles in the �ERKO are often characterized by a hypertrophied thecum, indicating stimulation by LH. Induction of thecal cell function by the chronically elevated serum LH is illustrated by the similarly elevated levels of serum androgens exhibited in the adult �ERKO female (Table 2). Histological analysis of ovaries from numerous �ERKO females has revealed no corpora lutea, indicating an inability of the follicles to spontaneously ovulate and differentiate. Anovulation in the adult �ERKO could not be rescued even after appropriate stimulation with exogenous gonadotropins (142), strongly indicating that infertility in the �ERKO female is due in large degree to alterations in the hormonal milieu and ovarian responsiveness. A phenotype of anovulation and follicular arrest is also observed in the ovaries of other murine models of gene disruption. The ovaries of mice lacking the actions of FSH, either due to targeted disruption of the gene for the FSH�subunit (241) or the FSH-receptor (242), exhibit follicles that grow up to but not beyond the preantral stage. Therefore, FSH action is critical to completion of the follicular phase of the ovarian cycle. However, �ERKO female mice possess serum FSH levels within the normal range (Table 2) and actually show elevated levels of FSH-receptor mRNA in the ovary (142). Furthermore, since FSH signaling has been proposed to be a critical factor in estradiol synthesis (243, 244), the extremely elevated levels of serum estradiol in the �ERKO female indicate that this pathway is intact in the granulosa cells. Interestingly, neither report of mice lacking FSH action mention any significant differences in ovarian estradiol synthesis but describe a much smaller and thinner uterus in the female (241, 242). It is possible that the estradiol present in these models lacking FSH action is synthesized by the ovarian thecum. Alternatively, the presence of compensatory mechanisms that have allowed for granulosa cell aromatase activity that is independent of FSH stimulation must also be considered. Progression of the follicle to the more mature antral stage is also arrested in mice after targeted disruption of the gene for IGF-I (245), cyclin-D2 (246), connexin-37 (247), activin type II receptor (248), and superoxide dismutase 1 (249), although low serum gonadotropin levels are thought to be the cause of this phenotype in the latter two models. Therefore, a definitive cause of the �ERKO ovarian phenotype, in addition to the loss of ER� action, was not obvious. In fact, some facets of ovarian physiology thought to be dependent on estrogen action, such as the attenuation of apoptosis and the induction of LH receptors in granulosa FIG. 4. Histology of a representative adult wild-type and �ERKO ovary. Shown are ovarian cross-sections from wild-type (a) and �ERKO (b) females at low magnification (13.2�). Note the presence of follicles at both the follicular and luteal (corpora lutea; CL) stages of the ovarian cycle in the wild-type ovary, whereas the �ERKO ovary is characterized by the presence of large, hemorrhagic, and cystic follicles, a sparse number of follicles at the early stages of proliferation, and a lack of corpora lutea. Panels c–d illustrate �ERKO ovarian tissue at high power magnification (132�): c, a healthy secondary follicle showing a normal oocyte and nucleolus; d, interface of two cystic follicles, demonstrating the heterogeneity in the number of granulosa cells that line the cysts, e.g., the left cyst has a single layer, whereas the right cyst has several layers of granulosa cells (indicated by arrows); e, hypertrophied thecal cells lining a hemorrhagic cyst in an �ERKO ovary. Scale bar � 0.5 �m.
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