Estrogen Receptor Null Mice - Endocrine Reviews

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June, 1999 ESTROGEN RECEPTOR NULL MICE 373 Nonetheless, the deciduomas induced in the �ERKO females are neither reduced in size nor appear less complex or differentiated than those observed in the wild type (183). It is also possible that a lack of ER� during development as well as adulthood has resulted in a uterus with a heightened tendency toward decidualization, caused by to the altered expression of other gene products. The complexity of the decidual process is illustrated by the several models that lack the ability to exhibit uterine decidualization, such as mice lacking leukemia-inhibitory factor (188), prostaglandin synthase-2 (189), and Hoxa-10 (190). Furthermore, a process thought to be critical to implantation is the acquired ability of portions of the uterine epithelium to self-destruct and become detached from the uterine wall, possibly clearing a route by which the underlying swelling endometrium can breach and provide a site for implantation (186, 191). Histological analysis of �ERKO uteri indicates a uterine epithelium that may be less healthy and more often exhibits sloughing compared with wild-type uteri. It is therefore possible that the inherently impaired luminal epithelium of the �ERKO female has resulted in a lowering of the threshold required to induce decidualization. B. Vagina The fully developed adult vagina serves as both a copulatory receptacle and a birth canal in the female and may be divided into two distinct sections, the upper vagina and lower vagina. The cranial end of the upper vagina is attached to the cervix and is derived from the Müllerian ducts during differentiation of the female tract (143). The lower vagina, which connects the tract to the vulva and external genitalia, is differentiated from the urogenital sinus (143). As shown in Fig. 3, the wild-type vagina is a highly sensitive estrogen target tissue, composed of an inner mucosal layer of stroma and overlying epithelia, a middle layer of muscularis, and an outer sheath of connective tissue. Detectable levels of ER� are present in both the stromal and epithelial compartments making up the mucosa of the duct (7). Estradiol exposure during the ovarian cycle induces a series of effects in the vaginal mucosa that are often used to estimate serum gonadal hormone levels and approximate the current stage in the estrous cycle (107). These changes in the mucosa include cytodifferentiation of the stromal cells and a rapid proliferation and differentiation of the epithelial cells, resulting in a stratified and cornified epithelial layer closest to the lumen (192). This process also involves the estrogen stimulation of a series of keratins (193, 194). As shown in Fig. 3, despite the chronically elevated levels of estradiol in the serum of �ERKO females, histological analysis consistently indicates a complete lack of vaginal estrogenization. A similar effect on the vaginal mucosa has been produced in mice (195, 196) and rats (197) after prolonged ovariectomy or treatments with the antiestrogens, ZM-189,154, EM-800, and tamoxifen. Administration of exogenous estradiol, DES, or hydroxytamoxifen to �ERKO mice results in no discernible vaginal response (153). In contrast, the vaginal mucosa of the �ERKO female appears to undergo the normal cyclic changes associated with ovarian steroidogenesis (Fig. 3) (47), strongly indicating that this is an ER�-mediated process. Buchanan et al. (198) have used the stromal-epithelial tissue recombinant scheme described above for uterine tissue to dissect the contributions of the different tissue compartments in the estradiol response of vaginal tissue as well. As in the uterus, these studies demonstrated that stromal ER�, but not epithelial ER�, are required for estradiol-induced epithelial proliferation in the mouse vagina (198). Similar to the observations in the uterine recombinants, both vaginal stromal and epithelial ER� were required for estradiol-induced stratification and cornification of the epithelium, including the induction of the gene for the secretory protein cytokeratin 10 (198). All recombinations involving tissue from the �ERKO vagina became atrophied, even in the presence of estradiol (198). C. Oviduct The mouse oviduct is a coiled tubular organ connecting the uterus to the ovarian bursa and derived from the Müllerian duct during fetal development of the female reproductive tract (143). It functions as a route for passage of sperm to the ovulated oocyte and the subsequently fertilized blastocyst to the uterus. In the CD-1 mouse, ER� immunoreactivity is easily detectable in both the stroma and the epithelium of the oviduct as early as day 2 of life (144). The levels of ER� immunoreactivity in the epithelium continue to rise and plateau at approximately day 15 and remain high throughout adulthood (144). Furthermore, Newbold et al. (199, 200) described the high degree of sensitivity of the fetal and neonatal oviduct to the detrimental effects of developmental DES exposure. During adulthood, the levels of ER� in the oviductal epithelium fluctuate during the ovarian cycle, reaching peak levels during the proliferative phase (201). The ovarian sex hormones found in high concentrations in the oviductal fluid are thought to play a role in ovum and zygote transport through the oviduct (201). However, studies using ovariectomized laboratory animals have produced conflicting results in terms of what this role may be, depending on the animal model and hormone dosing regimen used (202). Despite the apparent ontogeny of ER� in the developing and adult mouse oviduct, no gross phenotypes in the oviduct of �ERKO females have been observed. Similar to the uterus, the epithelium of the �ERKO oviduct usually appears healthy yet unstimulated, despite the chronically high levels of serum estradiol. Due to the inability of the �ERKO to ovulate, possible defects in the transport functions of the oviduct are not easily studied. Assays for ER� mRNA in the mouse oviduct detect little if any ER� transcripts. Accordingly, in �ERKO females there appears to be no obvious defects in the structure and function of the oviduct that impede fertility. D. Ovary In most mammals, differentiation of the bipotential fetal gonad to an ovary in the genotypic female occurs later in gestation than differentiation of the testis in the male (111). The factors involved in development and differentiation of the ovary are not well understood, although the process does not appear to be dependent on the presence of primordial germ cells (111). The appearance of follicles and the onset of
374 COUSE AND KORACH Vol. 20, No. 3 estrogen synthesis in the fetal gonad may be the first indication of differentiation to an ovary, although the secreted estrogens do not appear to be critical to development of the ductal structures of the female reproductive tract. Still, fetal ovarian estradiol may play a role in development of the ovary itself, as evidenced by the complete lack of ovaries in SF-1 knockout mice (137, 138). Furthermore, a recent study has demonstrated immunohistochemical detection of ER� and ER� in the neonatal rat ovary (103). Interestingly, a lack of ER� or ER� during development appears to have no gross effect on ovarian differentiation, since individual knockouts of both respective receptors possess normal ovaries at birth and during prenatal development (47, 142). A study of ovarian development in a double-knockout lacking both forms of ER will therefore prove interesting in the future. Still, distinct ovarian phenotypes become apparent in the adult �ERKO and �ERKO females, resulting in infertility and subfertility in each, respectively (46, 47). 1. Review of the physiology and function of the ovary. A brief description of ovarian morphology is necessary for a discussion of phenotypes that result from a lack of ER. The ovary may be conveniently divided into three broad functional units: the follicles, corpora lutea, and interstitial/stromal compartment (203, 204). All three possess the capacity to synthesize hormonal factors, especially steroids, in response to gonadotropins secreted from the anterior pituitary. The maturing follicle is a relatively ellipsoidal unit that can be further divided into three main cell types: the outermost thecal cells, which surround a single or multiple layers of granulosa cells, and together act to encase the germ cell (oocyte) at the approximate core. The overall size of the follicle and the number of cells composing the thecal and granulosa cell compartments are dependent on the stage of maturation (204). The corpora lutea are clearly defined and vascularized structures formed from the terminally differentiated thecal and granulosa cells that remain after ovulation (203). And finally, the interstitial and stromal tissue is composed of undifferentiated cells that may eventually be recruited for the thecal or granulosa units as well as dedifferentiated thecal and granulosa cells from past atretic follicles or regressed corpora lutea. This compartment also functions as the matrix within which the follicles are suspended (203). Ovarian function is often divided into two separate phases. The follicular phase refers to the period of follicle maturation and increased estradiol synthesis that leads up to and terminates with ovulation of the ovum. Ovulation marks the beginning of the luteal stage in which the developing corpora lutea secrete large amounts of progesterone as well as estradiol to allow for successful implantation of the blastocysts in the uterus. During the follicular stage of the ovarian cycle, the follicles may be categorized based on size, responsiveness to gonadotropins, and steroidogenic capabilities. These stages are most commonly referred to as the primordial, primary, secondary, tertiary or antral, atretic, and mature Graafian follicle (204). The primordial, or nongrowing follicles, are the most prevalent stage in the ovary at any one time and provide the pool from which follicles will be selected for maturation. These follicles consist of an oocyte arrested at the diplotene stage of the first meiotic division, surrounded by a single layer of cuboidal granulosa cells (204). Commencement of the follicular phase of the ovarian cycle involves the recruitment of primordial follicles to form the assembly of primary growing follicles to be prepared for ultimate ovulation. The factors required for this recruitment are not well understood. Henceforth, each stage is characterized by dramatic changes in the structure and functional capabilities of the follicle, which have been well characterized in several reviews (204–208). As the follicle progresses toward the secondary stage, rapid proliferation of the granulosa cells results in the formation of several concentric layers surrounding the oocyte (204). By this stage, stromal cells have differentiated to produce a defined stratum of thecal cells that encapsulate the granulosa cell/oocyte unit. A basement membrane acts to separate the heavily vascularized thecal layers from the granulosa cells and ovum. Since capillaries do not penetrate the basement membrane, the granulosa/oocyte compartment depends on the passive movement of hormonal factors through this extracellular structure (204). Oocyte and follicular growth are linear up to the tertiary stage, at which time the ooctye appears to reach a maximum size, while the follicle as a unit continues to enlarge. The tertiary follicle is characterized by a hypertrophied thecal layer, multiple layers of granulosa cells, and the appearance of an antrum, a space that separates the granulosa cells from the ooctye/cumulus complex. The process of follicular selection for ovulation, although not well understood, appears to occur at this stage of folliculogenesis, when several secondary-tertiary follicles will divert toward a pathway of atresia. Still under the influence of gonadotropins, the “selected” follicles will continue to enlarge, mostly due to increases in antrum size, to eventually reach the Graafian, or ovulatory stage. Follicular rupture and ovulation occur in response to a surge in gonadotropin levels, at which time cellular proliferation is ceased, and the remaining cells of the follicle terminally differentiate to form the corpus luteum (209). In consonance with its gametogenic function, the ovary fulfills a critical role as an endocrine organ, serving as the principal source of sex steroids in the female. Therefore, a normal functioning ovary is an essential prerequisite to the function and maintenance of the reproductive tract, mammary gland, and behavior of the female. The research efforts of several investigators during the past decades have generated the well accepted “two-cell, two-gonadotropin” model of ovarian estradiol synthesis. This model and the investigations leading to its description have been discussed in great detail in several recent reviews and therefore will be only summarized here (204, 206, 210). The two steroid-producing components of the maturing follicle are the thecal and granulosa cells, which predominantly produce androgens and estrogens, respectively (210). Ample evidence exists to indicate that thecal cells possess the full complement of steroidogenic enzymes necessary for estradiol synthesis. In contrast, estradiol synthesis by the granulosa cells is dependent on thecal-derived androgens as substrates for aromatization. The amount and activity of the expressed steroidogenic enzymes within the two cell types vary depending on the follicular stage, and thereby determine the predominant ste-
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