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D B Lubahn

Publications and source records attributed to D B Lubahn.

At least 55 records · Page 3Linked to original sources
PubMed1999

Cloning, sequencing, and localization of bovine estrogen receptor-beta within the ovarian follicle.

The potential role of estrogen receptor-beta (ERbeta) in normal ovarian folliculogenesis and in reproductive disorders such as ovarian follicular cysts has not been well defined. Therefore, we were interested in cloning, sequencing, and localizing ERbeta mRNA and protein within the bovine ovary. Bovine ERbeta (bERbeta) was amplified by reverse transcription-polymerase chain reaction (RT-PCR), then cloned and sequenced. Results showed that the open reading frame of bERbeta cDNA spanned 1584 nucleotides encoding a protein of 527 amino acids. The N-terminal region of bERbeta was found to be 80% homologous to human and mouse ERbeta and 79% homologous to rat ERbeta. Bovine ERbeta DNA-binding domain was 100% homologous to human, mouse, and rat ERbeta sequences. The C-terminal/ligand-binding domain of bERbeta was 89% homologous to human, 86% homologous to mouse, and 88% homologous to rat ERbeta. Human and bovine ERbeta amino acid sequences are similar in that their coding region extended farther 5' than initially reported for the published rat ERbeta sequence. Using in situ hybridization and immunohistochemistry, ERbeta mRNA and protein, respectively, were demonstrated to be present in granulosa cells of antral follicles in various stages of follicular growth. These findings suggest a role for bERbeta in ovarian follicular growth and maturation.

Amino Acid Sequence↗
PubMed1999

Tissue compartment-specific estrogen receptor-alpha participation in the mouse uterine epithelial secretory response.

17Beta-estradiol (E2) acts through the estrogen receptor (ER) to regulate uterine epithelial cell growth, proliferation, differentiation, and secretory protein production. We have previously shown that E2-induced uterine epithelial proliferation is mediated indirectly by ER alpha-positive stroma; epithelial ER alpha is neither necessary nor sufficient for E2-induced uterine epithelial mitogenesis. In the present study, we addressed the question of whether production of uterine epithelial secretory proteins and their messenger RNAs (mRNAs) requires ER alpha in stroma, epithelium, or both by analyzing tissue recombinations composed of uterine tissue from adult ER alpha knockout (ko) and neonatal BALB/c (wt) mice. Stroma (S) and epithelium (E) were separated by trypsinization, and four types of uterine tissue recombinants were prepared: wt-S + wt-E, wt-S + ko-E, ko-S + wt-E, and ko-S + ko-E. These tissue recombinants were grown as subrenal capsule grafts in intact female nude mice for 4 weeks, at which time the hosts were ovariectomized. To assess the production of secretory proteins and their mRNAs, 1 week after ovariectomy the hosts were given three daily injections of oil or E2 (100 ng), and then 24 h later the grafts were recovered and used for either ER or lactoferrin (LF) immunohistochemistry. To assess steady state mRNA levels by Northern blotting, hosts received one injection of oil or E2 24 h before harvest. ER immunohistochemistry was used to monitor the completeness of tissue separation. In wt-S + wt-E tissue recombinants from E2-treated hosts, the epithelium stained intensely for LF (an abundant E2-dependent uterine secretory protein), whereas similar tissue recombinants from oil-treated hosts showed minimal immunostaining. Conversely, LF immunostaining was minimal in wt-S + ko-E grafts from both oil- and E2-treated hosts. LF staining was also minimal in ko-S + ko-E and ko-S + wt-E tissue recombinants regardless of hormone treatment. For Northern analyses, the epithelial content of the tissue recombinants was monitored using the reference epithelial transcript, E-cadherin. While all tissue recombinant groups expressed E-cadherin mRNA, wt-S + wt-E tissue recombinants from E2-treated hosts produced a strong, single 2.6-kb band of LF mRNA. LF transcripts were minimal or absent in all other tissue recombinant types. Northern blotting results identical to those seen for LF were also observed for the uterine secretory protein complement component C3. Our data demonstrate that both stromal and epithelial ER alpha are required for the production of LF protein and of LF or C3 mRNAs in response to E2. Thus, in contrast to E2-induced epithelial mitogenesis, which requires only stromal ER alpha, both epithelial and stromal ER alpha are necessary for the production of E2-dependent epithelial secretory proteins.

Animals↗
PubMed1999

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.

Animals↗
PubMed1999

Uterine decidual response occurs in estrogen receptor-alpha-deficient mice.

Embryo-uterine interactions leading to the attachment reaction is followed by stromal cell proliferation and differentiation into decidual cells (decidualization) at the sites of blastocyst apposition. In rodents, decidualization is also induced by application of an artificial stimulus (intraluminal oil infusion) in a pseudopregnant uterus, or to one that has been appropriately prepared by exogenous progesterone (P4) and estrogen. The process of decidualization is under the control of these steroids in the presence of blastocysts or deciduogenic stimuli. Although it is well known that estrogen is required for the induction of progesterone receptors in the uterus, the functional importance of estrogen in the process of decidualization is poorly understood. To better understand the role of estrogenic actions in decidualization, we used wild-type and estrogen receptor-alpha knock-out (ERKO) mice for induction of decidualization employing a defined steroid hormonal treatment schedule. Our results demonstrate that P4 alone induces decidualization in ovariectomized wild-type or ERKO mice in response to intraluminal oil infusion in the absence of estrogen. A combined treatment of either estradiol-17beta (E2) or its catecholmetabolite 4-hydroxyestradiol-17beta(4-OH-E2) with P4 does not potentiate the decidual response produced by P4 treatment alone in either ovariectomized wild-type or ERKO mice. The induction of decidual response was associated with up-regulation of decidual cell marker genes, such as progesterone receptor, metallothionein-1, and cyclooxygenase-2. The results suggest that the stromal cell sensitivity to decidualization is critically dependent on P4-regulated events, and estrogenic induction of progesterone receptor via classical nuclear ER-alpha is not critical for this process.

Animals↗
PubMed1999

Methoxychlor stimulates estrogen-responsive messenger ribonucleic acids in mouse uterus through a non-estrogen receptor (non-ER) alpha and non-ER beta mechanism.

This study examined the effects of the xenoestrogen methoxychlor (Mxc) on messenger RNA (mRNA) concentrations of two estrogen-responsive uterine genes, lactoferrin (LF) and glucose-6-phosphate dehydrogenase (G6PD). Ovariectomized wild-type (WT) and estrogen receptor (ER) alpha-knockout (ER alphaKO) mice were treated with Mxc or estradiol-17beta (E2) to determine whether Mxc acts via pathways that involve ER alpha. In WT mice, both E2 and Mxc stimulated increases in uterine LF and G6PD mRNA concentrations in a dose-dependent manner. Competitive pretreatment with the pure antiestrogen ICI 182,780 dramatically reduced E2-stimulated increases in mRNA concentrations but had no effect on Mxc-induced effects. Competitive pretreatment with E2 had only a partially inhibitory effect on Mxc-induced responses. In the ER alphaKO mouse, E2 had little effect on uterine LF or G6PD mRNA concentrations, whereas Mxc stimulated marked increases in both LF and G6PD mRNAs. The Mxc-induced increases in LF and G6PD mRNAs in the ER alphaKO mouse were not suppressed by competitive pretreatment with either E2 or ICI 182,780. Fold increases in mRNA concentrations for both genes induced by Mxc were similar for WT and ER alphaKO mice. The results surprisingly indicate that a xenoestrogen, Mxc, can increase LF and G6PD mRNA concentrations by a mechanism that is not mediated through ER alpha or ER beta, and acts through another pathway.

Animals↗
PubMed1999

Steroid feedback on gonadotropin release and pituitary gonadotropin subunit mRNA in mice lacking a functional estrogen receptor alpha.

Steroid hormones regulate levels of gonadotropin mRNA in the pituitary, and gonadotropic hormones in plasma. To determine whether estrogen receptor alpha (ERalpha) mediates steroid negative feedback, wild type (WT) and estrogen receptor alpha knockout (ERalphaKO) mice of both sexes were gonadectomized and implanted with a Silastic capsule containing either estradiol (E2), dihydrotestosterone (DHT), testosterone, or a blank capsule. Ten days later, plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were measured. Pituitary mRNA levels of gonadotropin subunit (alpha, LHbeta, FSHbeta) and prolactin (PRL) were quantified. LH levels in gonad-intact ERalphaKO females were elevated, similar to values seen following gonadectomy. By contrast, serum LH concentrations in gonad-intact ERalphaKO males were low and rose following gonadectomy, suggesting androgen feedback. Estradiol treatment significantly decreased plasma LH in WT animals, but not in ERalphaKOs. In fact, in female ERalphaKOs, our dose of E2 increased plasma levels of LH as compared with untreated, ovariectomized ERalphaKOs. All the steroid treatments suppressed LH in WT animals whereas only DHT consistently suppressed LH concentrations in ERalphaKO mice. The postgonadectomy rise in plasma FSH was prevented by steroid treatments in WT females, but not in any of the other groups. Gonadotropin subunit and PRL mRNA responses to E2 treatment (both inhibitory and stimulatory) were absent in ERalphaKO mice, suggesting a critical role for ERalpha. Although E2 can exert negative feedback effects on LH release in both males and females by actions at the ERalpha, the androgen receptor plays the primary physiological role in the male mouse.

Animals↗
PubMed1998

Estrogen receptor mutations.

The purpose of this paper is to review potential novel functional pathways by which estradiol and estrogenic compounds elicit biological responses in mammals. We will limit our approach to those novel functions suggested by phenotypes associated with estrogen receptor-alpha (ER alpha) gene mutations and polymorphisms. The study of these pathways has been greatly aided by the availability of ER alpha-minus mice, which lack classic biological responses to estradiol. In addition, the availability of an ER alpha-minus human family, aromatase-minus human families, and in the near future an aromatase-minus mouse model will allow correlations of novel phenotypes with the lack of active ER alpha protein. The ER alpha-minus mice can potentially be used to characterize in depth novel clinical phenotypes that link the functions of estrogens with sexual maturation, cardiovascular disease, osteoporosis, diabetes, and cancer.

Animals↗
PubMed1998

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.

Androgen-Insensitivity Syndrome↗
PubMed1998

Role of stromal and epithelial estrogen receptors in vaginal epithelial proliferation, stratification, and cornification.

Estradiol 17-beta (E2) induces epithelial proliferation, stratification, and cornification in vaginal epithelium. Our aim was to determine the respective roles of epithelial and stromal estrogen receptor-alpha (ER alpha) in these E2-induced events. Vaginal epithelium (E) and stroma (S) from adult ER alpha knockout (ko) and wild-type (wt) neonatal Balb/c mice were enzymatically separated and used to produce four types of tissue recombinants in which epithelium, stroma, or both lack functional ER alpha. Tissue recombinants were grafted into female nude mice, which were subsequently ovariectomized and treated with oil or E2. In response to E2 treatment, grafts prepared with wt-S (wt-S + wt-E and wt-S + ko-E) showed similar large increases in epithelial labeling index, indicating that E2 stimulated epithelial proliferation despite a lack of epithelial ER alpha in wt-S + ko-E tissue recombinants. Conversely, in tissue recombinants prepared with ko-S (ko-S + wt-E and ko-S + ko-E), epithelial labeling index remained at baseline levels after E2 or oil treatment, even though epithelial ER alpha were detected in ko-S + wt-E grafts. Epithelial cornification was present in wt-S + wt-E grafts from E2-treated hosts, whereas epithelium in all other tissue recombinants failed to cornify. Grafts composed of wt-S + wt-E from E2-treated hosts had highly stratified epithelium, whereas epithelial thickness was reduced almost 60% in wt-S + ko-E tissue recombinants grown in E2-treated hosts and was atrophic in all other tissue recombinants. In addition, cytokeratin 10, a marker of epithelial differentiation, was strongly expressed in wt-S + wt-E tissue recombinants grown in E2-treated hosts but was markedly reduced or absent in all other tissue recombinants. These results indicate that E2-induced vaginal epithelial proliferation is mediated indirectly through stromal ER alpha, consistent with our recent findings in uterus. Conversely, both epithelial and stromal ER alpha are required for E2-induced cornification and normal epithelial stratification. These are the first known functions attributed to epithelial ER alpha in vivo and the first time any epithelial response to E2 has been shown to involve both stromal and epithelial ER alpha.

Animals↗
PubMed1998

Roles of estrogen receptor-alpha gene expression in reproduction-related behaviors in female mice.

The role of gene expression of the estrogen receptor-alpha form (ER alpha) in the regulation of female reproductive behavior was investigated in estrogen receptor knockout (ERKO) mice, deficient specifically for the ER alpha, but not the ER beta, gene. Estrogen- or estrogen- plus progesterone-treated gonadectomized ERKO mice did not show any lordosis response. Detailed behavioral analysis revealed that ERKO females were also deficient in sexual behavioral interactions preceding the lordosis response. They were extremely rejective toward attempted mounts by stud male mice, which could not show any intromissions. During resident-intruder aggression tests, gonadally intact ERKO females were more aggressive toward female intruder mice than wild-type (WT) mice. Gonadectomy did not influence the levels of aggressive behavior, and their genotype differences when mice were tested both before and after gonadectomy. However, when mice were tested after gonadectomy for the first time, very few ERKO mice showed aggression. In contrast to aggression, male-type sexual behavior shown by resident mice toward female intruder mice during aggression tests was not different between ERKO and WT mice and was completely abolished after gonadectomy of the resident mice. Finally, it was also found that ERKO females showed greatly reduced levels of parental behavior toward newborn pups placed in their home cage. These changes in parental behavior were not influenced by gonadectomy. ERKO females retrieved significantly fewer numbers of pups with longer latencies compared with wild-type (WT) or heterozygous (HZ) littermates when they were tested as gonadally intact or 20-65 days after gonadectomy. In addition, during parental behavior tests, a significantly higher percentage of ERKO mice exhibited infanticide compared with WT and HZ mice, which rarely showed infanticide. Taken together, these findings suggest that ER alpha gene expression plays a key role in female mice, not only for sexual behavior but also for other interrelated behaviors, such as parental and aggressive behaviors. In addition, persistence of genotype differences in parental and aggressive behavior after gonadectomy indicates that ER alpha activation during neural developmental processes may also be involved in the regulation of these behaviors.

Aggression↗
PubMed1998

Modifications of testosterone-dependent behaviors by estrogen receptor-alpha gene disruption in male mice.

The role of the a form of estrogen receptor (ER alpha) gene expression in the regulation of testosterone-dependent male reproductive behaviors was investigated using ER knockout mice (ERKO), which are specifically deficient in functional ER alpha, but not ER beta, gene expression. Previous studies in gonadally intact ERKO mice revealed that male aggressive behavior was greatly reduced by the lack of a functional ER alpha gene. In the present study the almost complete suppression of male-typical offensive attacks was further confirmed in ERKO mice that had been singly housed since weaning. Regarding aggression, it was also found that ER alpha gene disruption virtually abolished the propensity to initiate offensive attacks, even though ERKO mice could elicit attacks from resident C57BL/6J mice as wild-type (WT) and heterozygous littermates. Daily injection of testosterone propionate (TP) was completely ineffective in inducing aggressive behavior in gonadectomized ERKO mice, whereas it successfully restored aggression in WT mice. In contrast, male sexual behaviors, mounts and intromissions, were induced by daily injection of TP in both gonadectomized ERKO and WT mice. In addition to TP, dihydrotestosterone propionate (DHTP) was also effective in restoring mounts in ERKO mice, although DHTP was much more potent in WT mice than in ERKO mice. Neither TP nor DHTP, however, ever induced ejaculation in ERKO mice. These results together with previous findings in gonadally intact ERKO mice suggest that ER alpha may be responsible for the regulation by testosterone of consummatory, but not motivational, aspects of male sexual behavior. Finally, ERKO male mice retrieved newborn pups placed in their home cage with similar latencies to males of the two other genotypes. During parental behavior tests, however, a higher percentage of ERKO mice (70%) showed infanticide compared with WT mice (35%). The latter result was interpreted as showing that ER alpha activation by testosterone during the perinatal period may exert a suppressive effect on testosterone-inducible infanticide in adulthood. With respect to three major testosterone-dependent behavioral systems reflecting masculinization, these findings demonstrate three different types of effects due to ER alpha gene disruption.

Aggression↗
PubMed1998

Transcription and translation of estrogen receptor-beta in the male reproductive tract of estrogen receptor-alpha knock-out and wild-type mice.

Estrogen receptor-alpha (ERalpha) has been identified in the male reproductive tract, but the role of estrogen in the male has not been well characterized. In vivo mutations in ERalpha genes have demonstrated the necessity for ERalpha-mediated action in male fertility. We asked whether both ERbeta messenger RNA and protein were present in the male reproductive tract of wild-type and ERalpha knock-out (ERalpha KO) mice, and whether ERbeta could compensate for the lack of ERalpha in infertile male ERalpha KO mice. Immunohistochemical localization with both N- and C-terminal anti-ERbeta antibodies demonstrated that ERbeta is present in the Leydig cells of the testes and in the epithelium of both the efferent ductules and the initial segment of the epididymis. RT-PCR amplification was used to confirm ERbeta transcription in these tissues. In conclusion, we observed that ERbeta messenger RNA and protein continue to be expressed in the Leydig cells, elongated spermatids, efferent ductules, and the initial segment of the epididymides of ERalpha KO mice, but the presence of ERbeta is not able to compensate for the absence of ERalpha in male reproductive function.

Animals↗