The bi-modal effects of estradiol on gonadotropin synthesis and secretion in female mice are dependent on estrogen receptor-alpha.

Depending on the estrous/menstrual cycle stage in females, ovarian-derived estradiol (E(2)) exerts either a negative or a positive effect on the hypothalamic-pituitary axis to regulate the synthesis and secretion of pituitary gonadotropins, LH, and FSH. To study the role of estrogen receptor-alpha (ERalpha) mediating these effects, we assessed the relevant parameters in adult wild-type (WT) and ERalpha-null (alphaERKO) female mice in vivo and in primary pituitary cell cultures. The alphaERKO mice exhibited significantly higher plasma and pituitary LH levels relative to WT females despite possessing markedly high levels of circulating E(2).

Regulation of mouse renal CYP2J5 expression by sex hormones.

Mouse CYP2J5 is abundant in kidney and active in the metabolism of arachidonic acid to epoxyeicosatrienoic acids. Western blots of microsomes prepared from mouse kidneys demonstrate that after puberty, CYP2J5 protein is present at higher levels in male mice than in female mice. Northern analysis reveals that CYP2J5 transcripts are more abundant in adult male versus female kidneys, indicating that gender differences in renal CYP2J5 expression are regulated at a pretranslational level.

Molecular characterization and expression of vitellogenin receptor from white perch (Morone americana).

A full-length (4021 base pair [bp]) cDNA encoding a polypeptide (844 amino acids) with a predicted mass of 93 kDa and other characteristic structural features of a vertebrate vitellogenin receptor (VgR) was isolated from a white perch (Morone americana) ovarian cDNA library. Northern blotting performed using a specific digoxygenin-labeled VgR cDNA probe revealed a distinct approximately 4.1 kilobase (kb) hybridization signal in an mRNA preparation obtained from previtellogenic perch ovaries.

A unique application to the IACUC for studies of wild animals in or from natural settings.

The format of an application to the Institutional Animal Care and Use Committee (IACUC) frequently is designed to obtain detailed information from medical faculty proposing studies involving a precisely determined number of a few well-characterized species in a controlled laboratory setting. Unfortunately, these application formats typically are less than ideal for marine and field biologists attempting to propose studies of large populations of diverse organisms in a comparatively uncontrolled environment and somewhat unpredictable setting. Traditional IACUC applications rarely address topics of field capture, restraint, marking, animal care in the field, and release of animals back into the environment.

Requirement of estrogen receptor-alpha in insulin-like growth factor-1 (IGF-1)-induced uterine responses and in vivo evidence for IGF-1/estrogen receptor cross-talk.

In the uterus insulin-like growth factor-1 (IGF-1) signaling can be initiated by estradiol acting through its nuclear receptor (estrogen receptor (ER)) to stimulate the local synthesis of IGF-1. Conversely, in vitro studies have demonstrated that estradiol-independent ER transcriptional activity can be induced by IGF-1 signaling, providing evidence for a cross-talk mechanism between IGF-1 and ER. To investigate whether ER alpha is required for uterine responses to IGF-1 in vivo, both wild-type (WT) and ER alpha knockout (alpha ERKO) mice were administered IGF-1, and various uterine responses to IGF-1 were compared.

Induction of mammary gland development in estrogen receptor-alpha knockout mice.

Mammary glands from the estrogen receptor-a knockout (alphaERKO) mouse do not undergo ductal morphogenesis or alveolar development. Disrupted ERalpha signaling may result in reduced estrogen-responsive gene products in the mammary gland or reduced mammotropic hormones that contribute to the alphaERKO mammary phenotype. We report that circulating PRL is reduced in the female alphaERKO mouse.

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.

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.

Estrogen-dependent gene regulation by an oxidative metabolite of diethylstilbestrol, diethylstilbestrol-4',4"-quinone.

Diethylstilbestrol (DES) is a well-characterized carcinogen in humans and animals although its mechanisms of carcinogenicity are not yet known. While the estrogenic activity of DES is important, there is evidence that oxidative metabolism also plays an important role for its toxicity. DES is oxidatively metabolized in vivo and in vitro to a number of compounds including diethylstilbestrol-4',4"-quinone (DQ), an unstable and reactive intermediate, and Z,Z-dienestrol (ZZ-DIEN).

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.

Role of estrogen receptor-alpha in the anterior pituitary gland.

Targeted insertional disruption of the mouse estrogen receptor-alpha (ER alpha) gene has provided a genetic model in which to test hypotheses that estrogens exert important effects in development and homeostatic functions of the anterior pituitary gland, particularly in the lactotroph and gonadotroph cell types. Analysis of ER alpha gene-disrupted mice reveals a marked reduction in PRL mRNA and a decrease in lactotroph cell number, but normal specification of lactotroph cell phenotype. Gonadotropin mRNA levels in ER alpha gene-disrupted female mice are elevated, consistent with previously described transcriptional suppression of gonadotropin subunit gene expression in response to sustained administration of estrogen in wild type mice.

Developmental and physiological effects of estrogen receptor gene disruption in mice.

Disruption of the estrogen receptor (ER) gene in mice causes infertility in both sexes. Infertility in female ER knockout (ERKO) mice results from altered development of accessory sex structures, disrupted endocrine physiology, and disrupted gametogenesis. Male accessory sex structures appear relatively normal, with infertility stemming from altered sexual behaviors and disrupted gametogenesis.

Physiological coupling of growth factor and steroid receptor signaling pathways: estrogen receptor knockout mice lack estrogen-like response to epidermal growth factor.

Past studies have shown that epidermal growth factor (EGF) is able to mimic the uterotropic effects of estrogen in the rodent. These studies have suggested a "cross-talk" model in which EGF receptor (EGF-R) signaling results in activation of nuclear estrogen receptor (ER) and its target genes in an estrogen-independent manner. Furthermore, in vitro studies have indicated the requirement for ER in this mechanism.

LY191704 inhibits type I steroid 5 alpha-reductase in human scalp.

Conversion of testosterone to dihydrotestosterone (DHT) has been demonstrated to be catalyzed by two isoforms of steroid 5 alpha-reductase, designated types I and II. Although several classes of steroid-based inhibitors of the type II isoform have been identified, these agents have not demonstrated highly selective pharmacological activity against human type I 5 alpha-reductase. LY191704 is representative of a series of nonsteroidal agents that have potent [apparent inhibitory constant (Ki) = 11.

Estrogen receptor gene disruption: molecular characterization and experimental and clinical phenotypes.

The estrogen receptor (ER) is thought to play a crucial role in the regulation of many life processes, including development, reproduction and normal physiology. Because there have been no known mutations of the estrogen receptor in normal tissue of humans and animals, its presence and tissue distribution is thought to be essential for survival. Using the techniques of homologous recombination, we have disrupted the ER gene and have produced a line of transgenic mice possessing the altered ER gene (ERKO).

Analysis of transcription and estrogen insensitivity in the female mouse after targeted disruption of the estrogen receptor gene.

We employed homologous recombination in mouse embryonic stem cells to disrupt the estrogen receptor (ER) gene. Subsequently generated mice that are homozygous for the gene disruption, termed ERKO, possess no demonstrable wild-type ER by Western blot analysis. However, the presence of residual high affinity binding, as detected by [3H]estradiol binding assays and sucrose gradients in uterine extracts from ERKO females prompted further investigation of transcription and translation products from the disrupted ER gene.

Calcium regulation of androgen receptor expression in the human prostate cancer cell line LNCaP.

Elevation of intracellular calcium levels in the presence of normal androgen levels has been implicated in apoptotic prostate cell death. Since the androgen receptor (AR) plays a critical role in the regulation of growth and differentiation of the prostate, it was of interest to determine whether Ca2+ would affect the expression of androgen receptor messenger RNA (mRNA) and protein, thus affecting the ability of androgens to control prostate function. AR-positive human prostate cancer cells, LNCaP, were incubated with either the calcium ionophore A23187 or the intracellular endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin.

The mouse androgen receptor gene contains a second functional promoter which is regulated by dihydrotestosterone.

The androgen receptor (AR) is a developmental and tissue-specific transcription factor which is activated by binding testosterone or dihydrotestosterone. Several different methods of transcriptional regulation of the AR have been shown, including regulation by androgens, follicle-stimulating hormone, epidermal growth factor, and the cAMP pathway. In order to further characterize the transcriptional regulation of the AR, portions of the mouse androgen receptor (mAR) promoter were cloned into the promoterless pBLCAT3 vector and assayed for chloramphenicol acetyltransferase activity.

Antagonism of androgen action in prostate tumor cells by retinoic acid.

Recent studies have demonstrated that retinoic acid (RA) can repress the growth of human prostatic epithelial cells. Since the proliferation of prostate cells is highly dependent on androgen stimulation, presumably via its cognate receptor, we investigated the effects of RA on the expression of the androgen receptor and other androgen-regulated genes in the human prostatic adenocarcinoma cell line LNCaP. Using a radioligand binding assay, we found that androgen-binding activity was reduced 30-40% in cells treated with 10(-5) M RA plus 6 nM dihydrotestosterone (DHT), as compared to cells with the androgen alone.

The mouse androgen receptor is suppressed by the 5'-untranslated region of the gene.

The androgen receptor (AR) mediates the biological functions of androgens and is essential for normal growth and differentiation of urogenital organs as well as initiation and maintenance of spermatogenesis. Withdrawal of androgens by castration or other methods has been shown to cause a marked, although often temporary, regression of many prostate cancers. In order to gain a better understanding of the transcriptional regulation of the AR, a series of truncation mutants derived from the 5'-region of the mouse AR (mAR) were inserted into the promoter-less plasmid pBLCAT3 and transiently expressed in the mouse alpha T3-1 and GT1-7 cell lines.

The androgen receptor in the testicular feminized (Tfm) mouse may be a product of internal translation initiation.

Androgen insensitivity in the testicular feminized (Tfm) mouse is caused by frame-shift mutation in the androgen receptor (AR) mRNA, which results in a stop codon in the amino terminus. Despite this mutation, a smaller sized protein corresponding to the DNA- and steroid-binding domain of the AR can be synthesized from the cloned Tfm AR cDNA by in vitro translation. The Tfm AR construct was demonstrated to express a protein capable of binding androgen with an affinity similar to the cloned wild-type AR.

Molecular mechanisms of androgen action.

Androgens directly regulate a vast number of physiological events. These direct androgen effects are mediated by a nuclear receptor that exhibits four major functions or activities: steroid binding, DNA binding, transactivation, and nuclear localization. The SBD consists of a hydrophobic pocket of amino acids that exhibits high-affinity, androgen-specific binding.

Regulation of the 5'-flanking region of the mouse androgen receptor gene by cAMP and androgen.

Both androgens and cAMP-mediated hormones are known to regulate expression of the androgen receptor (AR) gene. In order to determine whether these effects occur at the transcriptional level, transfection studies were conducted with a 1.5-kilobase fragment of the 5'-flanking region of the mouse AR gene coupled to a chloramphenicol acetyltransferase (CAT) reporter gene.

Effects of progesterone and dihydrotestosterone on stimulation of androgen-dependent sex behavior, accessory sex structures, and in vitro binding characteristics of cytosolic androgen receptors in male whiptail lizards (Cnemidophorus inornatus).

Progestins often act as potent antiandrogens in male birds and mammals. Experiments with lizards find that progestins can both inhibit (when given in high dosages) or stimulate (when given in low dosages) male-typical sex behavior in gonadectomized individuals. This study shows that in the little striped whiptail lizard exogenous progesterone (P) facilitates androgen-dependent sex behaviors in males yet fails to stimulate seasonal activation of androgen-dependent accessory sex structures.