17beta-estradiol induces apoptosis in the developing rodent prostate independently of ERalpha or ERbeta.

Estrogens induce both proliferative and antiproliferative responses in the prostate gland. To date, antiproliferative effects of estrogens are generally considered to be due to systemic antiandrogenic actions. However, estrogen action mediated through estrogen receptor (ER) beta was recently suggested as another mechanism of induction of apoptosis in the prostate. This study aimed to explore the hypothesis that the antiproliferative effects of estrogen are directly mediated through ERbeta using a prostate organ culture system. We previously reported effects of 17beta-estradiol (E2) using rat ventral prostate (VP) tissues, and adapted the system for culturing mouse tissues. In both rat and mouse models, estrogen-induced apoptosis was detected that was spatially and regionally localized to the epithelium of the distal tips. Using organ cultures of alphaER knockout (alphaERKO) and betaERKO prostates, we failed to demonstrate that apoptosis induced by E2 was mediated through either receptor subtype. Activation of ER-selective ligands (ERalpha, propyl pyrazole triol, ERbeta, diaryl-proprionitrile, and 5alpha-androstane-3beta,17beta-diol) in organ culture experiments failed to induce apoptosis, as did the membrane impermeable conjugate E2:BSA, discounting the possibility of nongenomic effects. Consequently, E2 regulation of androgen receptor (AR) expression was examined and, in the presence of nanomolar testosterone levels, E2 caused a specific reduction in AR protein expression in wild-type, alphaERKO, and betaERKO mice, particularly in the distal region where apoptosis was detected. This down-regulation of AR protein provides a possible mechanism for the proapoptotic action of E2 that is independent of ERs or nongenomic effects.

Estrogen imprinting of the developing prostate gland is mediated through stromal estrogen receptor alpha: studies with alphaERKO and betaERKO mice.

Neonatal exposure of rodents to high doses of estrogen permanently imprints the growth and function of the prostate and predisposes this gland to hyperplasia and severe dysplasia analogous to prostatic intraepithelial neoplasia with aging. Because the rodent prostate gland expresses estrogen receptor (ER)-alpha within a subpopulation of stromal cells and ERbeta within epithelial cells, the present study was undertaken to determine the specific ER(s) involved in mediating prostatic developmental estrogenization. Wild-type (WT) mice, homozygous mutant ER (ERKO) alpha -/- mice, and betaERKO -/- mice were injected with 2 microg of diethylstilbestrol (DES) or oil (controls) on days 1, 3, and 5 of life. Reproductive tracts were excised on days 5 or 10 (prepubertal), day 30 (pubertal), day 90 (young adult), or with aging at 6, 12, and 18 months of age. Prostate complexes were microdissected and examined histologically for prostatic lesions and markers of estrogenization. Immunocytochemistry was used to examine expression of androgen receptor, ERalpha, ERbeta, cytokeratin 14 (basal cells), cytokeratin 18 (luminal cells), and dorsolateral protein over time in the treated mice. In WT-DES mice, developmental estrogenization of the prostate was observed at all of the time points as compared with WT-oil mice. These prostatic imprints included transient up-regulation of ERalpha, down-regulation of androgen receptor, decreased ERbeta levels in adult prostate epithelium, lack of DLP secretory protein, and a continuous layer of basal cells lining the ducts. With aging, epithelial dysplasia and inflammatory cell infiltrate were observed in the ventral and dorsolateral prostate lobes. In contrast, the prostates of alphaERKO mice exhibited no response to neonatal DES either immediately after exposure or throughout life up to 18 months of age. Furthermore, neonatal DES treatment of betaERKO mice resulted in a prostatic response similar to that observed in WT animals. The present findings indicate that ERalpha is the dominant ER form mediating the developmental estrogenization of the prostate gland. If epithelial ERbeta is involved in some component of estrogen imprinting, its role would be considered minor and would require the presence of ERalpha expression in the prostatic stromal cells.

Estrogen receptor-alpha knockout mice exhibit resistance to the developmental effects of neonatal diethylstilbestrol exposure on the female reproductive tract.

Data indicate that estrogen-dependent and -independent pathways are involved in the teratogenic/carcinogenic syndrome that follows developmental exposure to 17beta-estradiol or diethylstilbestrol (DES), a synthetic estrogen. However, the exact role and extent to which each pathway contributes to the resulting pathology remain unknown. We employed the alphaERKO mouse, which lacks estrogen receptor-alpha (ERalpha), to discern the role of ERalpha and estrogen signaling in mediating the effects of neonatal DES exposure. The alphaERKO provides the potential to expose DES actions mediated by the second known ER, ERbeta, and those that are ER-independent. Wild-type and alphaERKO females were treated with vehicle or DES (2 microg/pup/day for Days 1-5) and terminated after 5 days and 2, 4, 8, 12, and 20 months for biochemical and histomorphological analyses. Assays for uterine expression of the genes Hoxa10, Hoxa11, and Wnt7a shortly after treatment indicated significant decreases in DES-treated wild-type but no effect in the alphaERKO. In contrast, the DES effect on uterine expression of Wnt4 and Wnt5a was preserved in both genotypes, suggesting a developmental role for ERbeta. Adult alphaERKO mice exhibited complete resistance to the chronic effects of neonatal DES exposure exhibited in treated wild-type animals, including atrophy, decreased weight, smooth muscle disorganization, and epithelial squamous metaplasia in the uterus; proliferative lesions of the oviduct; and persistent vaginal cornification. Therefore, the lack of DES effects on gene expression and tissue differentiation in the alphaERKO provides unequivocal evidence of an obligatory role for ERalpha in mediating the detrimental actions of neonatal DES exposure in the murine reproductive tract.

Contrasting phenotypes in reproductive tissues of female estrogen receptor null mice.

The estrogen receptor (ER) exists in two known forms, ERalpha and ERbeta, and acts as a ligand-inducible transcription factor to fulfill critical roles in reproductive physiology. Although in vitro studies suggest the ERs may play redundant roles, a dissimilar tissue distribution indicates otherwise. Therefore, to gain insight into the role of each ER form, individual lines of mice lacking each respective receptor, as well as mice lacking both ER forms, were generated. alphaERKO and betaERKO female mice possess a normally developed reproductive tract and maintain expression of the opposite ER. The alphaERKO female is infertile and exhibits a hypoplastic uterus that is refractory to estrogens. The ovaries of the alphaERKO female are consistently polycystic and lack indications of spontaneous ovulation. In contrast, the betaERKO female exhibits a hormonally responsive uterus and grossly normal ovaries, but is subfertile in terms of the frequency and size of litters. Immature females of both ERKO lines successfully ovulate viable ova when superovulated with exogenous gonadotropins, yet the average yield of ooctyes is reduced. Mice lacking both known ER forms (alphabetaERKO) are infertile, possess the expected reproductive tract structures, but exhibit a remarkably distinct ovarian phenotype characterized by postnatal loss of oocytes and redifferentiation of the remaining somatic cells to Sertoli-like cells. This "sex-reversal" in the alphabetaERKO ovary is accompanied by the ectopic expression of testis-specific genes, for example, Sox9 and sulfatedglycoprotein-2.

17beta-estradiol and ICI-182780 regulate the hair follicle cycle in mice through an estrogen receptor-alpha pathway.

Estradiol (E(2)) applied topically twice weekly to mouse skin at doses as low as 1 nmol inhibited hair growth by blocking the transition of the hair follicle from the resting phase (telogen) to the growth phase (anagen). In contrast, application of

Expression of estrogen receptor beta is developmentally regulated in reproductive tissues of male and female mice.

By the use of ribonuclease protection assay (RPA) combined with immunohistochemical techniques, the expression of estrogen receptor (ER) alpha and ERbeta was mapped in the developing gonads and reproductive tracts of male and female mice from fetal day 14 to postnatal day 26 (PND 26). This study was designed to determine the pattern of expression of both ER subtypes in specific tissue compartments during development. In ovaries, ERalpha mRNA was detected at all ages examined; ERbeta mRNA was seen as early as PND 1, and its expression increased with age. Immunolocalization showed ERbeta in differentiating granulosa cells of the ovary, whereas ERalpha was predominantly seen in interstitial cells. The remainder of the female reproductive tract showed ERalpha mRNA at all ages examined with little or no significant levels of ERbeta, except on PND 1 when a low level of message appeared. In males, ERalpha and ERbeta mRNA were detected in the fetal testis; however, ERbeta gradually increased until PND 5 and subsequently diminished to undetectable levels by PND 26. Immunolocalization showed ERalpha in the interstitial compartment of the testis, whereas ERbeta was seen predominantly in developing spermatogonia. The remainder of the male reproductive tract showed varying amounts of both receptors by RPA and immunostaining throughout development. These studies provide information useful in studying the role of both ER subtypes in normal differentiation, and they provide indications of differential tissue expression during development.

Estrogen receptor transcription and transactivation: Estrogen receptor knockout mice: what their phenotypes reveal about mechanisms of estrogen action.

Natural, synthetic and environmental estrogens have numerous effects on the development and physiology of mammals. Estrogen is primarily known for its role in the development and functioning of the female reproductive system. However, roles for estrogen in male fertility, bone, the circulatory system and immune system have been established by clinical observations regarding sex differences in pathologies, as well as observations following menopause or castration. The primary mechanism of estrogen action is via binding and modulation of activity of the estrogen receptors (ERs), which are ligand-dependent nuclear transcription factors. ERs are found in highest levels in female tissues critical to reproduction, including the ovaries, uterus, cervix, mammary glands and pituitary gland. Since other affected tissues have extremely low levels of ER, indirect effects of estrogen, for example induction of pituitary hormones that affect the bone, have been proposed. The development of transgenic mouse models that lack either estrogen or ER have proven to be valuable tools in defining the mechanisms by which estrogen exerts its effects in various systems. The aim of this article is to review the mouse models with disrupted estrogen signaling and describe the associated phenotypes.

Prevention of the polycystic ovarian phenotype and characterization of ovulatory capacity in the estrogen receptor-alpha knockout mouse.

Ovarian-derived estradiol plays a critical endocrine role in the regulation of gonadotropin synthesis and secretion from the hypothalamic-pituitary axis. In turn, several para/autocrine effects of estrogen within the ovary are known, including increased ovarian weight, stimulation of granulosa cell growth, augmentation of FSH action, and attenuation of apoptosis. The estrogen receptor-alpha (ERalpha) is present in all three components of the hypothalamic-pituitary-ovarian axis of the mouse. In contrast, estrogen receptor-beta (ERbeta) is easily detectable in ovarian granulosa cells but is low to absent in the pituitary of the adult mouse. This distinct expression pattern for the two ERs suggests the presence of separate roles for each in the regulation of ovarian function. Herein, we definitively show that a lack of ERalpha in the hypothalamic-pituitary axis of the ERalpha-knockout (alphaERKO) mouse results in chronic elevation of serum LH and is the primary cause of the ovarian phenotype of polycystic follicles and anovulation. Prolonged treatment with a GnRH antagonist reduced serum LH levels and prevented the alphaERKO cystic ovarian phenotype. To investigate a direct role for ERalpha within the ovary, immature alphaERKO females were stimulated to ovulate with exogenous gonadotropins. Ovulatory capacity in the immature alphaERKO female was reduced compared with age-matched wild-type (14.5+/-2.9 vs. 40.6+/-2.6 oocytes/animal, respectively); however, oocytes collected from the alphaERKO were able to undergo successful in vitro fertilization. A similar discrepancy in oocyte yield was observed after superovulation of peripubertal (42 days) wild-type and alphaERKO females. In addition, ovaries from immature superovulated alphaERKO females possessed several ovulatory but unruptured follicles. Investigations of the possible reasons for the reduced number of ovulations in the alphaERKO included ribonuclease protection assays to assess the mRNA levels of several markers of follicular maturation and ovulation, including ERbeta, LH-receptor, cyclin-D2, P450-side chain cleavage enzyme, prostaglandin synthase-2, and progesterone receptor. No marked differences in the expression pattern for these mRNAs during the superovulation regimen were observed in the immature alphaERKO ovary compared with that of the wild-type. Serum progesterone levels just before ovulation were slightly lower in the alphaERKO compared with wild-type. These studies indicate that treatment of alphaERKO females with a GnRH antagonist decreased the serum LH levels to within the wild-type range and concurrently prevented development of the characteristic ovarian phenotype of cystic and hemorrhagic follicles. Furthermore, a lack of functional ERalpha within the ovary had no effect on the regulation of several genes required for follicular maturation and ovulation. However, the reduced numbers of ovulations following the administration of exogenous gonadotropins in the alphaERKO suggests an intraovarian role for ERalpha in follicular development and ovulation.

Postnatal sex reversal of the ovaries in mice lacking estrogen receptors alpha and beta.

Mice lacking estrogen receptors alpha and beta were generated to clarify the roles of each receptor in the physiology of estrogen target tissues. Both sexes of alphabeta estrogen receptor knockout (alphabetaERKO) mutants exhibit normal reproductive tract development but are infertile. Ovaries of adult alphabetaERKO females exhibit follicle transdifferentiation to structures resembling seminiferous tubules of the testis, including Sertoli-like cells and expression of Müllerian inhibiting substance, sulfated glycoprotein-2, and Sox9. Therefore, loss of both receptors leads to an ovarian phenotype that is distinct from that of the individual ERKO mutants, which indicates that both receptors are required for the maintenance of germ and somatic cells in the postnatal ovary.

Reproductive phenotypes in the estrogen receptor-alpha knockout mouse.

The generation and initial characterizations of the ER alpha KO and has proved exciting, however, it is worth noting that this model is relatively new to the field. These mice will undoubtedly prove invaluable to future studies of steroid hormones in normal development and function. The ER alpha KO mouse has been and continues to be utilized to further study the role of estrogen action in the cardiovascular system. bone physiology, behavior, the immune system, neurophysiology, and adipogenesis. Furthermore, studies of the documented "non-genomic" effects of estradiol and progesterone in the brain as well as ligand-independent actions involving cross-talk with other signaling systems will be advanced with further investigations using the ER alpha KO mouse. And finally, the ER alpha KO in combination with the recently developed ER beta KO mouse will prove invaluable in distinguishing the different roles that may exist between the two receptors in the estrogen signaling system.

Estrogen receptor null mice: what have we learned and where will they lead us?

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 alpha ERKO mice continue to satisfy the confirmatory role of a knockout quite well. As summarized in Table 4, the phenotypes observed in the alpha ERKO due to estrogen insensitivity have definitively illustrated several roles that were previously believed to be dependent on functional ER alpha, 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 alpha ERKO must begin with the observation that generation of an animal lacking a functional ER alpha gene was successful and produced animals of both sexes that exhibit a life span comparable to wild-type. The successful generation of beta 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 alpha ERKO. In support of this is our recent successful generation of double knockout, or alpha beta 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 alpha ERKO, were quite surprising. In turn, certain estrogen pathways in the alpha 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. [ABSTRACT TRUNCATED]

Targeted disruption of the estrogen receptor-alpha gene in female mice: characterization of ovarian responses and phenotype in the adult.

Targeted disruption of the mouse estrogen receptor-alpha gene (estrogen receptor-alpha knockout; ERKO) results in a highly novel ovarian phenotype in the adult. The ERKO mouse model was used to characterize ER alpha-dependent processes in the ovary. Visualization of the ovaries of 10-, 20-, and 50-day-old wild-type (WT) and ERKO mice showed that the ERKO phenotype developed between 20 and 50 days of age. Developmental progression through the primordial, primary, and antral follicle stages appeared normal, but functional maturation of preovulatory follicles was arrested resulting in atresia or in anovulatory follicles, which in many cases formed large, hemorrhagic cysts. Corpora lutea were absent, which also indicates that the normal biochemical and mechanical processes that accomplish ovulation were compromised. Northern and ribonuclease protection analyses indicated that ERKO ovary FSH receptor (FSHR) messenger RNA (mRNA) expression was approximately 4-fold greater than in WT controls. Ovarian LH receptor (LHR) mRNA expression was also higher in the ERKO animals. Cellular localization studies by in situ hybridization analysis of ERKO ovaries showed a high level of LHR mRNA expression in the granulosa and thecal layers of virtually all the antral follicles. Ribonuclease protection analyses showed that ovarian progesterone receptor and androgen receptor mRNA expression were similar in the two groups. These results indicated that ER alpha action was not a prerequisite for LHR mRNA expression by thecal or granulosa cells or for ovarian expression of progesterone receptor mRNA. Ovarian estrogen receptor beta (ER beta) was detected immunohistochemically, was sharply compartmentalized to the granulosa cells, and was expressed approximately equally in the ERKO animals and the WT controls. In contrast, ER alpha staining was present in the thecal cells but not the granulosa cells of the WT animals. The summary findings indicate that in the adult the major cause of the ERKO phenotype is high circulating LH interacting with functional LHR of the theca and granulosa cells. These features result in a failure of the normal maturational events leading to successful ovulation and luteinization and presumably involve both hypothalamic-pituitary and intraovarian mechanisms dependent upon ER alpha action. The presence of ER beta in the granulosa cells did not rescue the phenotype of the ovary.

A mouse mammary tumor virus-Wnt-1 transgene induces mammary gland hyperplasia and tumorigenesis in mice lacking estrogen receptor-alpha.

Estrogens have important functions in mammary gland development and carcinogenesis. To better define these roles, we have used two previously characterized lines of genetically altered mice: estrogen receptor-alpha (ER alpha) knockout (ERKO) mice, which lack the gene encoding ER alpha, and mouse mammary virus tumor (MMTV)-Wnt-1 transgenic mice (Wnt-1 TG), which develop mammary hyperplasia and neoplasia due to ectopic production of the Wnt-1 secretory glycoprotein. We have crossed these lines to ascertain the effects of ER alpha deficiency on mammary gland development and carcinogenesis in mice expressing the Wnt-1 transgene. Introduction of the Wnt-1 transgene into the ERKO background stimulates proliferation of alveolar-like epithelium, indicating that Wnt-1 protein can promote mitogenesis in the absence of an ER alpha-mediated response. The hyperplastic glandular tissue remains confined to the nipple region, implying that the requirement for ER alpha in ductal expansion is not overcome by ectopic Wnt-1. Tumors were detected in virgin ERKO females expressing the Wnt-1 transgene at an average age (48 weeks) that is twice that seen in virgin Wnt-1 TG mice (24 weeks) competent to produce ER alpha. Prepubertal ovariectomy of Wnt-1 TG mice also extended tumor latency to 42 weeks. However, pregnancy did not appear to accelerate the appearance of tumors in Wnt-1 TG mice, and tumor growth rates were not measurably affected by late ovariectomy. Small hyperplastic mammary glands were observed in Wnt-1 TG males, regardless of ER alpha gene status; the glands were similar in appearance to those found in ERKO/Wnt-1 TG females. Mammary tumors also occurred in Wnt-1 TG males; latency tended to be longer in the heterozygous ER alpha and ERKO males (86 to 100 weeks) than in wild-type ER alpha mice (ca. 75 weeks). We conclude that ectopic expression of the Wnt-1 proto-oncogene can induce mammary hyperplasia and tumorigenesis in the absence of ER alpha in female and male mice. The delayed time of tumor appearance may depend on the number of cells at risk of secondary events in the hyperplastic glands, on the carcinogenesis-promoting effects of ER alpha signaling, or on both.

Generation and reproductive phenotypes of mice lacking estrogen receptor beta.

Estrogens influence the differentiation and maintenance of reproductive tissues and affect lipid metabolism and bone remodeling. Two estrogen receptors (ERs) have been identified to date, ERalpha and ERbeta. We previously generated and studied knockout mice lacking estrogen receptor alpha and reported severe reproductive and behavioral phenotypes including complete infertility of both male and female mice and absence of breast tissue development. Here we describe the generation of mice lacking estrogen receptor beta (ERbeta -/-) by insertion of a neomycin resistance gene into exon 3 of the coding gene by using homologous recombination in embryonic stem cells. Mice lacking this receptor develop normally and are indistinguishable grossly and histologically as young adults from their littermates. RNA analysis and immunocytochemistry show that tissues from ERbeta -/- mice lack normal ERbeta RNA and protein. Breeding experiments with young, sexually mature females show that they are fertile and exhibit normal sexual behavior, but have fewer and smaller litters than wild-type mice. Superovulation experiments indicate that this reduction in fertility is the result of reduced ovarian efficiency. The mutant females have normal breast development and lactate normally. Young, sexually mature male mice show no overt abnormalities and reproduce normally. Older mutant males display signs of prostate and bladder hyperplasia. Our results indicate that ERbeta is essential for normal ovulation efficiency but is not essential for female or male sexual differentiation, fertility, or lactation. Future experiments are required to determine the role of ERbeta in bone and cardiovascular homeostasis.

Effects of castration and chronic steroid treatments on hypothalamic gonadotropin-releasing hormone content and pituitary gonadotropins in male wild-type and estrogen receptor-alpha knockout mice.

Testicular androgens are integral components of the hormonal feedback loops that regulate circulating levels of LHbeta and FSH. The sites of feedback include hypothalamic areas regulating GnRH neurons and pituitary gonadotropes. To better define the roles of androgen receptor (AR), estrogen receptor-alpha (ERalpha), and estrogen receptor-beta (ERbeta) in mediating feedback effects of sex steroids on reproductive neuroendocrine function, we have determined the effects of castration and steroid replacement therapy on hypothalamic GnRH content, pituitary LHbeta and FSHbeta messenger RNA (mRNA) levels, and serum gonadotropins in male wild-type (WT) and estrogen receptor-alpha knockout (ERKO) mice. Hypothalami from intact WT and ERKO males contained similar amounts of GnRH, whereas castration significantly reduced GnRH contents in both genotypes. Replacement therapy with estradiol (E2), testosterone (T), or dihydrotestosterone (DHT) restored hypothalamic GnRH content in castrated (CAST) WT mice; only the androgens were effective in CAST ERKOs. Analyses of pituitary function revealed that LHbeta mRNA and serum LHbeta levels in intact ERKOs were 2-fold higher than those in intact WT males. Castration increased levels of LHbeta mRNA (1.5- to 2-fold) and serum LHbeta (4- to 5-fold) in both genotypes. Both E2 and T treatments significantly suppressed LHbeta mRNA and serum LH levels in CAST WT males. However, E2 was completely ineffective, and T was only partially effective in suppressing these two indexes in the CAST ERKO males. DHT treatments stimulated a 50% increase in LHbeta mRNA and serum LH levels in WT males, whereas serum LH was significantly suppressed in DHT-treated ERKO males. Although the pituitaries from intact ERKO males contained similar amounts of FSHbeta mRNA, serum FSH levels were 20% higher than those in the intact WT males. Castration increased FSHbeta mRNA levels only in WT males, but significantly increased serum FSH levels in both genotypes. Both E2 and T treatments significantly suppressed serum FSH in CAST WT males, whereas only E2 suppressed FSHbeta mRNA. DHT treatments of CAST WT mice stimulated a small increase in serum FSH, but failed to alter FSHbeta mRNA levels. None of the steroid treatments exerted any significant effect on FSHbeta mRNA or serum FSH levels in CAST ERKOs. These data suggest that hypothalamic GnRH contents can be maintained solely through AR signaling pathways. However, normal regulation of gonadotrope function requires aromatization of T and activation of ERalpha signaling pathways in the gonadotrope. In addition, serum FSH levels in male ERKOs appear to be regulated largely by nonsteroidal testicular factors such as inhibin. Finally, these data suggest that hypothalamic ERbeta may not be involved in mediating the negative feedback effects of T on serum LH and FSH in male mice.

Exploring the role of sex steroids through studies of receptor deficient mice.

Decades of study have described a number roles fulfilled by the steroid hormones and their respective receptors in sexual differentiation and development, reproductive function and behavior, and more recently in the function and maintenance of non-reproductive organ systems, such as skeletal muscle, bone and coronary tissues. The biological effects of the steroid hormones are believed to be mediated in part by specific receptor proteins that demonstrated great specificity for their respective steroid ligands. Much of the experimental research of the functions of the sex steroid receptors has depended upon in vitro systems as well as in vivo methods that require surgical castration or the pharmacological administration of hormone antagonists. However, recently developed techniques that allow for manipulation of the mouse genome have been utilized to generate transgenic animals that lack functional estrogen or progesterone receptors. These transgenic animals, combined with the naturally existing Tfm mice which lack functional androgen receptor, now provide in vivo models for further study of the various actions of the sex steroids and their receptors. This review attempts to describe and compare the various phenotypes that result in each of these lines of mice, with emphasis on the development and function of the reproductive systems as well as reproductive behavior.

The role of estrogen receptors and androgen receptors in sex steroid regulation of B lymphopoiesis.

Several observations suggest that sex steroids might participate in steady state regulation of B lymphopoiesis. B cell precursors decline dramatically in bone marrow of pregnant or estrogen-treated mice. Reciprocally, the same cell populations are increased in hypogonadal mice or male castrates. Estrogen treatment of hypogonadal mice reduced precursors to normal. However, questions remain about which hormones and receptors are the most important. Furthermore, these observations need to be reconciled with advances regarding new sex steroid receptors. We have now characterized B lymphopoiesis in androgen receptor-deficient testicular feminization (Tfm) mice. Testicular feminization mice had substantially elevated numbers of B cell precursors in the bone marrow and B cells in the spleen as compared with wild-type mice. The importance of one estrogen receptor (ER alpha) was evaluated in gene-targeted mice, and B cell precursors were found to be within the normal range. Our previous studies indicated that hormone receptors in stromal cells may be important for estrogen-mediated suppression of B lymphopoiesis. We now show that estrogen-mediated inhibition of B cell precursor expansion in culture was blocked by a specific estrogen receptor antagonist (ICI 182,780). Stromal cells derived from ER alpha-targeted bone marrow were fully estrogen responsive. RT-PCR analyses of these stromal cells revealed splice-variant transcripts of ER alpha, as well as message for a recently discovered estrogen-binding receptor, ER beta. Thus, androgens may normally inhibit B lymphopoiesis through the androgen receptor, whereas estrogens might utilize one or more receptors to achieve the same physiologic response.

Tissue distribution and quantitative analysis of estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta) messenger ribonucleic acid in the wild-type and ERalpha-knockout mouse.

Until recently, only a single type of estrogen receptor (ER) was thought to exist and mediate the genomic effects of the hormone 17beta-estradiol in mammalian tissues. However, the cloning of a gene encoding a second type of ER, termed ERbeta, from the mouse, rat, and human has prompted a reevaluation of the estrogen signaling system. Based on in vitro studies, the ERbeta protein binds estradiol with an affinity similar to that of the classical ER (now referred to as ERalpha) and is able to mediate the effects of estradiol in transfected mammalian cell lines. Essential to further investigations of the possible physiological roles of ERbeta, and its possible interactions with ERalpha, are data on the tissue distribution of the two ER types. Herein, we have described the optimization and use of an RNase protection assay able to detect and distinguish messenger RNA (mRNA) transcripts from both the ERalpha and ERbeta genes in the mouse. Because this assay is directly quantitative, a comparison of the levels of expression within various tissues was possible. In addition, the effect of disruption of the ERalpha gene on the expression of the ERbeta gene was also investigated using the ERalpha-knockout (ERKO) mouse. Transcripts encoding ERalpha were detected in all the wild-type tissues assayed from both sexes. In the female reproductive tract, the highest expression of ERbeta mRNA was observed in the ovary and showed great variation among individual animals; detectable levels were observed in the uterus and oviduct, whereas mammary tissue was negative. In the male reproductive tract, significant expression of ERbeta was seen in the prostate and epididymis, whereas the testes were negative. In other tissues of both sexes, the hypothalamus and lung were clearly positive for both ERalpha and ERbeta mRNA. The ERKO mice demonstrated slightly reduced levels of ERbeta mRNA in the ovary, prostate, and epididymis. These data, in combination with the several described phenotypes in both sexes of the ERKO mouse, suggest that the biological functions of the ERbeta protein may be dependent on the presence of ERalpha in certain cell types and tissues. Further characterization of the physiological phenotypes in the ERKO mice may elucidate possible ERbeta specific actions.