Estrogen Receptor Null Mice - Endocrine Reviews

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June, 1999 ESTROGEN RECEPTOR NULL MICE 379 FIG. 5. Histology of representative adult wild-type and �ERKO ovary. Shown are ovarian cross-sections from adult wild-type (a) and �ERKO (c) females and those from immature wild-type (b) and �ERKO (d) females after superovulation treatment at low power magnification (13.2�). Note the presence of follicles at various stages of the follicular phase in both the wild-type (a) and �ERKO (c) ovaries from the adult females, illustrating relatively little observable difference between the two genotypes. However, upon superovulation, a distinct phenotype becomes apparent in the �ERKO. Comparison of the superovulated wild-type (b) with the similarly treated �ERKO (d), indicates the presence of multiple corpora lutea (CL) in the wild-type whereas only two CL are obvious in the �ERKO. Most notable are the multiple numbers of unruptured ovulatory follicles present in the superovulated �ERKO ovary (indicated by arrows). High-power magnification (132�) of the adult �ERKO ovary illustrates follicles at progressive stages of maturation (e) and a healthy tertiary follicle showing an oocyte with nucleolus (f). High-power magnification (132�) of the superovulated immature �ERKO ovary illustrates a typical unruptured ovulatory follicle possessing several layers of granulosa cells and a centrally located oocyte (g), and a corpora lutea (h) indicating the successful luteinization and terminal differentiation of the follicle that occurs after ovulation. [Panels a–d reproduced with permission from Ref. 47.] Scale bar � 1 �m. tract of �ERKO females suggest that induction of the P450 arom gene by FSH in the granulosa cells appears intact. However, investigation of the expression of the above as well as other granulosa cell components is required to determine the cause of the ovulatory phenotype of the �ERKO female. To gain further insight into the ovarian phenotype of the �ERKO female, immature knockout and wild-type mice were stimulated to ovulate by administration of superphysiological levels of gonadotropins (PMSG and hCG) (47). After several independent trials, it became obvious that the ovulatory capacity of the �ERKO female was dramatically reduced, yielding an average of 6 (�1.5) oocytes per female vs. 33 (�4.8) and 52
380 COUSE AND KORACH Vol. 20, No. 3 (�5.7) oocytes per female in the wild-type and heterozygous animals, respectively (Table 3) (47). Additionally, the cumulus mass that surrounded the ovulated follicles from the �ERKO females was consistently composed of a decreased number of cells and a lessened integrity when compared with ova yielded from wild-type controls. Most interesting was the histology of the ovaries from the superovulated �ERKO females, which indicated the presence of numerous preovulatory but unruptured follicles (Fig. 5). It therefore appeared that the follicles of the �ERKO ovary were able to respond to the proliferative effects of PMSG in terms of increased size and antrum formation. However, a severe deficit in the response to the gonadotropin surge (hCG), required to induce luteinization and rupture of the follicle, was obvious in the �ERKO. A small number of the selected follicles were able to be expelled, as evidenced by the presence of ova in the oviduct and of corpora lutea in the corresponding �ERKO ovary. Therefore, a lack of ER� resulted in a drastic reduction in ovulatory capacity, yet with incomplete penetrance. Until �ERKO females are treated and tested in a similar manner to the �ERKO studies described, it will be difficult to determine the precise role for each ER in the ovary. The observation of numerous unruptured Graafian follicles in the ovaries of superovulated �ERKO females is strikingly similar to the phenotype reported for mice possessing a targeted disruption of the cyclin-D2 gene (246). Cyclin-D2 is a positive regulator of cell cycle progression that is highly expressed in granulosa cells (reviewed in Ref. 258). Robker and Richards (259) demonstrated strong up-regulation of the cyclin-D2 gene by estradiol in rat granulosa cells and suggested that this protein may be a downstream mediator of the synergistic actions of FSH and estradiol that result in increased granulosa cell numbers in the maturing follicle. The dramatic increases in cyclin-D2 induced by estradiol are evident in vivo at both the mRNA and protein levels in the rat. Furthermore, assays on primary granulosa cell cultures from the rat indicate that estradiol stimulation of the cyclin-D2 gene can be inhibited by the estrogen antagonist, ICI-164,384, strongly suggesting that it is an ER-mediated process (259). Therefore, given that ER� is the predominant form of ER in the granulosa cells, disruption of the ER� gene may likely result in significant deficits in cyclin-D2 expression in the granulosa cells of the growing follicles in the �ERKO female. However, FSH is also able to stimulate increases in cyclin-D2, although the temporal pattern of regulation by FSH is distinct from that elicited by estradiol (259). Nonetheless, it is possible that FSH action in the follicles of the �ERKO ovary have provided for some degree of cyclin-D2 expression and thereby may explain the incomplete penetrance of the �ERKO ovarian phenotype. The �ERKO ovarian phenotype that becomes apparent after superovulation is also similar to that reported for knockout models of the genes for the PR (PRKO) (44) and prostaglandin synthase-2 (189). The dramatic increases in both PR (260) and prostaglandin synthase-2 (261) in the granulosa cells of the ovulatory follicle shortly after the gonadotropin surge have been well documented. Furthermore, the lack of follicular rupture in the respective knockout models supports a critical role for each of these components in ovulation (reviewed in Ref. 262). Lydon et al. (44) reported infertility in the PRKO and a consistent inability of superphysiological doses of hCG to induce follicular rupture. Although regulation of the PR gene is strongly influenced by estradiol in the uterus (see Section III.A), sufficient evidence exists to indicate that this may not be the case in the ovary. For example, although the wild-type ovary possesses extremely high intraovarian levels of estradiol and the presence of ER�, levels of PR mRNA and protein remain at a modest basal level in granulosa cells of the pre- and antral follicle (260). However, within 4–6 h after the gonadotropin surge, transcription of the PR gene has peaked at levels several fold that before the surge, only to return to near-basal levels within 20 h (260). The mechanism of this strong and transient induction of the PR gene by LH is known to include significant increases in intracellular cAMP, but may also involve phosphorylation of ER� and/or a coactivator, which then combine to act in a synergistic nature (262). Therefore, the elevation in PR levels in the granulosa cells of the ovulatory follicle that is critical to follicular rupture may be attenuated in the �ERKO ovary. Another possibility for a lack of spontaneous ovulation in the �ERKO female may be not intaovarian in nature, but rather due to altered gonadotropin synthesis and secretion from the hypothalamic-pituitary axis. The sex steroids play an important role as a positive regulator of the preovulatory surge (reviewed in Refs. 263 and 264). Although the exact mechanism of action by which estrogens may be involved is not well defined, studies have shown that estradiol can induce GnRH release from the hypothalamus as well as cause increases in the level of GnRH receptors in the anterior pituitary (263). The ER� may be the predominant form of ER in the pituitary of the adult female mouse (93); however, both ER� and ER� have been detected in various regions of the hypothalamus (88, 97, 265). Preliminary data in the �ERKO female indicate that tonic levels of serum LH are within the normal range. However, a lack of hypothalamic ER� may have reduced the potential for positive regulation by estradiol in the hypothalamic-pituitary axis and thereby may result in a reduction in the frequency and/or amplitude of the preovulatory gonadotropin surge. Nonetheless, the results of the superovulation studies described above, in which an artificial bolus of gonadotropin is administered to induce ovulation, indicate a severe phenotype that can be localized to the ovary of the �ERKO female. IV. Mammary Gland In mammals, the mammary gland is essentially undeveloped at birth and does not undergo full growth until the completion of puberty and, in fact, remains undifferentiated until pregnancy and lactation. Development of the mammary gland may be divided into five distinct stages: embryonic and fetal, prepubertal, pubertal, sexually mature adult, and pregnancy/lactation (reviewed in Ref. 266). The influential factors involved at each stage differ in both type and magnitude. Although the developmental factors involved during the embryonic and fetal stages of the female mammary gland are poorly understood, estrogen action does not appear to be essential (266). However, studies have shown that the fetal and neonatal mammary gland of rodents is
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Estrogen Receptor Null Mice - Endocrine Reviews

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