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The ciliary beat frequency (CBF) of guinea-pig fimbria during the ovarian cycle was measured by video microscopy using a high-speed camera (500 Hz). In the follicular phase, with increasing concentrations of beta-oestradiol ([betaE(2)]) and a low concentration of progesterone ([PRG]), CBF increased from 13.5 to 16 Hz. In the ovulatory phase, with further increase of [betaE(2)], CBF decreased gradually from 16 to 13.5 Hz. In the early luteal phase, with low [PRG] and [betaE(2)], CBF increased to 17 Hz; however, in the middle luteal phase, with increasing [PRG], CBF decreased (12 Hz), and in the late luteal phase, with decreasing [PRG], CBF increased to 15 Hz. Then, in the resting phase, with low [betaE(2)] and [PRG], CBF decreased immediately to 14 Hz. The CBF of the fimbria was measured in guinea-pigs treated with beta-oestradiol benzoate (betaE(2)B) or medroxyprogesterone (mPRG). A low dose of betaE(2)B increased CBF to 14.5 Hz, whereas a high dose decreased it to 11 Hz. A betaE(2) receptor blocker, ICI-182,780, abolished the betaE(2)B-induced CBF changes and maintained CBF at 12.0 Hz. Medroxyprogesterone decreased CBF to 12.5 Hz, and mifepristone (a PRG receptor blocker) abolished the mPRG-induced CBF decrease and maintained CBF at 15 Hz. The addition of both blockers increased CBF to 18 Hz, suggesting that activation of betaE(2) or PRG receptors decreases the CBF of the fimbria. In conclusion, a moderate [betaE(2)] increase maintains a high CBF (15.5 Hz) in the follicular phase, and then further [betaE(2)] increase decreases CBF to 13.5 Hz in the ovulatory phase. In the early and late luteal phase, low [betaE(2)] and [PRG] increase CBF to 17 and 15 Hz, respectively, and in the middle luteal phase a high [PRG] decreases CBF (to 12 Hz). Thus, the CBF of the fimbria was controlled by signals via betaE(2) and PRG receptors in guinea-pigs.

The orphan G protein coupled receptor, GPR30, has the characteristics of a high affinity, specific estrogen membrane receptor on Atlantic croaker oocytes and mediates estrogen inhibition of oocyte maturation in this perciform fish. In order to determine the broad applicability of these findings to other teleosts, similar experiments were conducted in a cyprinid fish, zebrafish, in the present study. GPR30 mRNA expression was detected in zebrafish oocytes but not in the ovarian follicular cells. Both spontaneous and 17, 20beta-dihyroxy-4-pregnen-3-one (DHP)-induced maturation of follicle-enclosed zebrafish oocytes was significantly decreased when they were incubated with either estradiol-17beta, or the GPR30 agonists, ICI 182 780 and tamoxifen, or with the GPR30 specific agonist G-1. On the other hand spontaneous oocyte maturation increased two-fold when zebrafish ovarian follicles were incubated with an aromatase inhibitor, ATD. Moreover, the stimulatory effects of ATD on germinal vesicle breakdown (GVBD) were partially reversed by co-treatment with 100 nM of E2 or G-1. These results suggest that endogenous estrogens acting through GPR30 are involved in maintaining meiotic arrest of zebrafish oocytes.

Estradiol is the most potent and ubiquitous member of a class of steroid hormones called estrogens. Fetuses and newborns are exposed to estradiol derived from their mother, their own gonads, and synthesized locally in their brains. Receptors for estradiol are nuclear transcription factors that regulate gene expression but also have actions at the membrane, including activation of signal transduction pathways. The developing brain expresses high levels of receptors for estradiol. The actions of estradiol on developing brain are generally permanent and range from establishment of sex differences to pervasive trophic and neuroprotective effects. Cellular end points mediated by estradiol include the following: 1) apoptosis, with estradiol preventing it in some regions but promoting it in others; 2) synaptogenesis, again estradiol promotes in some regions and inhibits in others; and 3) morphometry of neurons and astrocytes. Estradiol also impacts cellular physiology by modulating calcium handling, immediate-early-gene expression, and kinase activity. The specific mechanisms of estradiol action permanently impacting the brain are regionally specific and often involve neuronal/glial cross-talk. The introduction of endocrine disrupting compounds into the environment that mimic or alter the actions of estradiol has generated considerable concern, and the developing brain is a particularly sensitive target. Prostaglandins, glutamate, GABA, granulin, and focal adhesion kinase are among the signaling molecules co-opted by estradiol to differentiate male from female brains, but much remains to be learned. Only by understanding completely the mechanisms and impact of estradiol action on the developing brain can we also understand when these processes go awry.

There is an urgent need to identify and develop a new generation of therapeutic agents and systemic therapies targeting the estradiol (E2)/estrogen receptor (ER) signaling in breast cancer. In this regard, new information on the mechanisms of E2/ER function and/or cross talk with other prosurvival cascades should provide the basis for the development of other ideal anti-E2 therapies with the intent to enhance clinical efficacy, reduce side effects or both. Our very recent assessment of the mechanisms by which cancer-associated increased lipogenesis and its inhibition alters the E2/ER signaling discovered that fatty acid synthase (FASN), the enzyme catalyzing the terminal steps in the de novo biosynthesis of long-chain fatty acids, differentially modulates the state of sensitivity of breast and endometrial cancer cells to E2-stimulated ER transcriptional activation and E2-dependent cell growth and survival: 1) pharmacological inhibition of FASN activity induced a dramatic augmentation of E2-stimulated ER-driven gene transcription, whereas interference (RNAi)-mediated silencing of FAS gene expression drastically lowered E2 requirements for optimal activation of ER transcriptional activation in breast cancer cells; conversely, pharmacological and RNAi-induced inhibition of FASN worked as an antagonist of E2- and tamoxifen-dependent ER transcriptional activity in endometrial adenocarcinoma cells; 2) pharmacological and RNAi-induced inhibition of FASN synergistically enhanced E2-mediated down-regulation of ER protein and mRNA expression in breast cancer cells, whereas specific FASN blockade resulted in a marked down-regulation of E2-stimulated ER expression in endometrial cancer cells; and 3) FASN inhibition decreased cell proliferation and cell viability by promoting apoptosis in hormone-dependent breast and endometrial cancer cells. In this review we propose that, through a complex mechanism involving the regulation of MAPK/ER cross talk as well as critical E2-related proteins including the Her-2/neu (erbB-2) oncogene and the cyclin-dependent kinase inhibitors p21(WAF1/CIP1) and p27(Kip1), a previously unrevealed connection exists between FASN and the genomic and nongenomic ER activities in breast and endometrial cancer cells. From a clinical perspective, we suggest that if chemically stable FASN inhibitors or cell-selective systems able to deliver RNAi targeting FASN gene demonstrate systemic anticancer effects of FASN inhibition in vivo, additional preclinical studies to characterize their anti-breast cancer actions should be of great interest as the specific blockade of FASN activity may also provide a protective means against endometrial carcinoma associated with tamoxifen-based breast cancer therapy.

Classical action of steroid hormones, called genomic, includes binding to their intracellular receptor, require hours or days to occur and require transcriptional effects with subsequent modulation of protein expression. Some of the biological effects induced by steroids, and mainly by sex steroids, take place within seconds or few minutes, time far too fast to be due to the genomic changes. The rapid, nongenomic action of estradiol are attributed to membrane action, probably through variety of proteins present in cell membrane. The rapid effects of steroid hormones are manifold, ranging from activation of protein and tyrosine kinases, G proteins, and modulation of ion channels. The nongenomic way of action includes also non-direct control of processes of transcription and gene expression. There are at least three different way to interact with cell membrane. Steroids may change membrane fluidity, without binding to any known protein or receptor. Another way is allosteric modulation of non-specific for steroid hormones receptors, or structural and enzymatic protein present in cell membrane. Evidence suggests that the classical steroid receptors can be localized at the plasma membrane, triggering signals typical for G-proteins coupled receptors. Physiological significance of nongenomic action of steroids needs to be elucidated.

The extensive literature and the work from our laboratory illustrate the large number of complex processes affected by estrogen that might contribute to the striking ability of 17beta-estradiol (E2) and its derivatives to inhibit clinical and histological signs of experimental autoimmune encephalomyelitis (EAE) in mice. These effects require sustained exposure to relatively low doses of exogenous hormone and offer better protection when initiated prior to induction of EAE. However, oral ethinyl estradiol (EE) and fluasterone, which lacks estrogenic side effects, could partially reverse clinical EAE when given after the onset of disease. The three main areas discussed in this review include E2-mediated inhibition of encephalitogenic T cells, inhibition of cell migration into central nervous system tissue, and neuroprotective effects that promote axon and myelin survival. E2 effects on EAE were mediated through Esr1 (alpha receptor for E2) but not Esr2 (beta receptor for E2), as were its antiinflammatory and neuroprotective effects. A novel finding is that E2 up-regulated the expression of Foxp3 and CTLA-4 that contribute to the activity of CD4+CD25+ Treg cells. The protective effects of E2 in EAE suggest its use as therapy for MS, although the risk of cardiovascular disease may complicate treatment in postmenopausal women. This risk could be minimized by using subpregnancy levels of exogenous E2 that produced synergistic effects when used in combination another immunoregulatory therapy. Alternatively, one might envision using EE or fluasterone metabolites alone or in combination therapies in both male and female MS patients.

All mammalian uteri contain endometrial glands that synthesize or transport and secrete substances essential for survival and development of the conceptus (embryo/fetus and associated extraembryonic membranes). In rodents, uterine secretory products of the endometrial glands are unequivocally required for establishment of uterine receptivity and conceptus implantation. Analyses of the ovine uterine gland knockout model support a primary role for endometrial glands and, by default, their secretions in peri-implantation conceptus survival and development. Uterine adenogenesis is the process whereby endometrial glands develop. In humans, this process begins in the fetus, continues postnatally, and is completed during puberty. In contrast, endometrial adenogenesis is primarily a postnatal event in sheep, pigs, and rodents. Typically, endometrial adenogenesis involves differentiation and budding of glandular epithelium from luminal epithelium, followed by invagination and extensive tubular coiling and branching morphogenesis throughout the uterine stroma to the myometrium. This process requires site-specific alterations in cell proliferation and extracellular matrix (ECM) remodeling as well as paracrine cell-cell and cell-ECM interactions that support the actions of specific hormones and growth factors. Studies of uterine development in neonatal ungulates implicate prolactin, estradiol-17 beta, and their receptors in mechanisms regulating endometrial adenogenesis. These same hormones appear to regulate endometrial gland morphogenesis in menstruating primates and humans during reconstruction of the functionalis from the basalis endometrium after menses. In sheep and pigs, extensive endometrial gland hyperplasia and hypertrophy occur during gestation, presumably to provide increasing histotrophic support for conceptus growth and development. In the rabbit, sheep, and pig, a servomechanism is proposed to regulate endometrial gland development and differentiated function during pregnancy that involves sequential actions of ovarian steroid hormones, pregnancy recognition signals, and lactogenic hormones from the pituitary or placenta. That disruption of uterine development during critical organizational periods can alter the functional capacity and embryotrophic potential of the adult uterus reinforces the importance of understanding the developmental biology of uterine glands. Unexplained high rates of peri-implantation embryonic loss in humans and livestock may reflect defects in endometrial gland morphogenesis due to genetic errors, epigenetic influences of endocrine disruptors, and pathological lesions.

Estradiol (E(2))-signaling is widely considered to be exclusively mediated through the transcription-regulating intracellular estrogen receptor (ER) alpha and ERbeta. The aim of this study was to investigate transcription-independent E(2)-signaling in mouse IC-21 macrophages. E(2) and E(2)-BSA induce a rapid rise in the intracellular free Ca(2+) concentration ([Ca(2+)](i)) of Fura-2 loaded IC-21 cells as examined by spectrofluorometry. These changes in [Ca(2+)](i) can be inhibited by pertussis toxin, but not by the ER-blockers tamoxifen and raloxifene. The E(2)-signaling initiated at the plasma membrane is mediated through neither ERalpha nor ERbeta, but rather through a novel G protein-coupled membrane E(2)-receptor as revealed by RT-PCR, flow cytometry, and confocal laser scanning microscopy. A special feature of this E(2)-receptor is its sequestration upon agonist stimulation. Sequestration depends on energy and temperature, and it proceeds through a clathrin- and caveolin-independent pathway.

The mechanism by which estradiol (E2) acts on cell proliferation is still unclear. In this paper, we report the results of a series of experiments in an attempt to elucidate the effector pathway(s) involved in coupling the E2 receptors binding to cellular growth response in leiomyoma cells (LSMC). Under conditions of E2-dependent growth, E2 treatment of LSMC triggers rapid and transient activation of the MAP-kinase pathway. Interestingly, we demonstrate that the early downstream signal transduction events determined by E2-stimulation in quiescent LSMC, including the rapid protein tyrosine phosphorylation of a subset of intracellular proteins, such GAP, PI-3-K, and PLCgamma, and the concomitant activation of ancillary protein kinases, are related to E2-induced PDGF secretion. Moreover, we identify the PDGF, alone or in association with other growth factors, as the main growth factor involved in the proliferation response of LSMC to E2 stimulation. The addition of neutralizing antibodies anti-PDGF was able to inhibit the mitogenic activity present in LSMC conditioned media samples. On the other hand, E2 did not affect the constitutive expression as well as the ligand affinity of PDGF receptors on LSMC plasmamembrane. Cell treatment with the antiestrogen ICI 182780 correlate both with a perturbation of E2-induced transductional circuit and with the disappearance of the mitogenic factor, PDGF, in LSMC conditioned media; the latter therefore, represents the main autocrine mediator of cell growth modulation, upregulated by E2 and down-regulated by antiestrogenic compound. Our experiments suggest that growth factor secretion is an initial and integral part of the signaling events mediated by the estradiol receptors, not related, at least in part, to E2 transcriptional modulation., (Copyright 2001 Wiley-Liss, Inc.)

Although estrogen is known to induce new bone formation in the long bones of female mice, this response is only thought to occur following administration of high doses, suggesting that it may not be mediated by a conventional estrogen receptor. To address this question further, we first examined the stereospecificity of this response by comparing the potency of 17beta-estradiol (E(2)) in stimulating cancellous bone formation at the proximal tibial metaphysis of intact female mice with that of the relatively inactive stereoisomer, 17alpha-estradiol (alphaE(2)). We found that E(2) was significantly more potent than alphaE(2), as assessed by histomorphometry. To provide further evidence for an estrogen-receptor-mediated process, we examined whether E(2)-induced osteogenesis in intact female mice could be inhibited by the estrogen receptor antagonist, ICI 182,780 (ICI). Although ICI itself had no effect on histomorphometric indices of the proximal tibial metaphysis when given alone, it significantly inhibited the osteogenic response to E(2). Finally, we examined the dose dependency of E(2)-induced osteogenesis at the proximal tibial metaphysis in intact mice. We found that E(2) stimulated cancellous bone formation in a dose-dependent manner over a wide dose range (i. e., 1-4000 microg/kg per day), with significant increases observed at doses of 4 microg/kg per day and beyond. Our results raise the possibility that estrogen-induced osteogenesis in the mouse represents an estrogen-receptor-mediated response that is not confined solely to supraphysiological estrogen levels.

We tested the hypothesis that estrogen acutely stimulates constitutive NO synthase (cNOS) activity in human peripheral monocytes by acting on an estrogen surface receptor. NO release was measured in real time with an amperometric probe. 17beta-estradiol exposure to monocytes stimulated NO release within seconds in a concentration-dependent manner, whereas 17alpha-estradiol had no effect. 17beta-estradiol conjugated to BSA (E2-BSA) also stimulated NO release, suggesting mediation by a membrane surface receptor. Tamoxifen, an estrogen receptor inhibitor, antagonized the action of both 17beta-estradiol and E2-BSA, whereas ICI 182,780, a selective inhibitor of the nuclear estrogen receptor, had no effect. We further showed, using a dual emission microfluorometry in a calcium-free medium, that the 17beta-estradiol-stimulated release of monocyte NO was dependent on the initial stimulation of intracellular calcium transients in a tamoxifen-sensitive process. Leeching out the intracellular calcium stores abolished the effect of 17beta-estradiol on NO release. RT-PCR analysis of RNA obtained from the cells revealed a strong estrogen receptor-alpha amplification signal and a weak beta signal. Taken together, a physiological dose of estrogen acutely stimulates NO release from human monocytes via the activation of an estrogen surface receptor that is coupled to increases in intracellular calcium.

Estradiol receptors are regarded to predict a likely success of hormonal therapeutic efforts and the prognosis of breast cancer patients. But today its prognostic importance is controversial, discussed as either reflecting intrinsic property of the tumor tissue or better therapeutic accessibility of receptor positive tumors. Moreover, the most important clinical prognosticators--tumor size and axillary lymph node involvement do not seem to be related to the estradiol receptor status. In our investigation, the length of disease free interval is similar in estradiol receptor positive and negative patients and in all sites of distant metastases, but it is significantly reduced if more than 4 axillary lymph nodes are involved. Post recurrence survival is significantly longer in estradiol receptor positive than negative patients and also in patients treated by tamoxifen containing therapies. Its length is independent of the number of axillary lymph node metastases and the type of distant metastases, with a tendency to be longer in estradiol receptor positive than negative patients. In addition, the overall survival is longer for estradiol receptor positive than negative patients and becomes reduced with more than 4 axillary lymph node metastases. Frequency of deaths in estradiol receptor positive patients is half that of negative subjects. Furthermore, the length of overall survival is independent on the type of distant metastases, with tendency to be longer in estradiol receptor positive than negative patients. Longest overall survival could be observed for estradiol receptor positive patients who got therapy regimens containing tamoxifen. The weak prognostic advantages of estradiol receptor positive patients are interpreted by estradiol receptors as intrinsic parameters of breast cancer tissue characterizing more its biological behavior than therapeutic accessibility.

The ovarian steroid hormones estradiol and progesterone exert their effect by interacting with their intracellular receptors, which, after ligand binding translocate to the nucleus and bind to the promoter regions of target genes. The consequence is a change in the transcription rate of the target genes, followed by a change in production of the corresponding proteins. Target genes of the sexual steroid hormones include cytokines and growth factors, among them CSF-1, TGF-beta and LIF. The rhythm and activity of steroidogenesis, receptor modulation and transcription are reflected by cycle-specific proliferation and differentiation processes in the endometrium. Quantitative and/or qualitative molecular endocrinology is of increasing interest for better definition of morphological changes, although, as yet, the pathological laboratory test is of much less practical consequence than a suspicious vaginal sonography. In spite of the high standard of ultrasound techniques, however, most cases with slightly increased endometrial thickness show histologically benign changes of the endometrium rather than endometrial precancer or cancer. This is especially true for perimenopausal women with no other clinical findings. Yet, the cancer risk is increased in women under tamoxifen therapy. Hence, as a rule, these cases, when endometrial thickness exceeds 5 mm, need a diagnostic biopsy or abrasio.

Impaired insulin secretion is a hallmark in both type I and type II diabetic individuals. Whereas type I (insulin-dependent diabetes mellitus) implies ss-cell destruction, type II (non-insulin dependent diabetes mellitus), responsible for 75% of diabetic syndromes, involves diminished glucose-dependent secretion of insulin from pancreatic beta-cells. Although a clear demonstration of a direct effect of 17beta-estradiol on the pancreatic ss-cell is lacking, an in vivo insulinotropic effect has been suggested. In this report we describe the effects of 17beta-estradiol in mouse pancreatic ss-cells. 17beta-Estradiol, at physiological concentrations, closes K(ATP) channels, which are also targets for antidiabetic sulfonylureas, in a rapid and reversible manner. Furthermore, in synergy with glucose, 17beta-estradiol depolarizes the plasma membrane, eliciting electrical activity and intracellular calcium signals, which in turn enhance insulin secretion. These effects occur through a receptor located at the plasma membrane, distinct from the classic cytosolic estrogen receptor. Specific competitive binding and localization of 17beta-estradiol receptors at the plasma membrane was demonstrated using confocal reflective microscopy and immunocytochemistry. Gaining deeper knowledge of the effect induced by 17beta-estradiol may be important in order to better understand the hormonal regulation of insulin secretion and for the treatment of NIDDM. receptor.

In previous studies, we have shown that the baroreflex control of heart rate is significantly attenuated in females compared with age-matched males. This study investigated the role of estrogen in the modulation of baroreflex function in conscious unrestrained rats. Baroreflex-mediated decreases in heart rate in response to increments in blood pressure evoked by phenylephrine were evaluated in conscious freely moving male and female Sprague-Dawley rats as well as in ovariectomized rats. The effect of a 2-day 17 beta-estradiol (50 micrograms.kg-1.day-1, s.c.) or vehicle treatment on baroreflex sensitivity was investigated in ovariectomized rats. Intravenous bolus doses of phenylephrine (1-16 micrograms/kg) elicited dose-dependent pressor and bradycardic responses in all groups of rats. Regression analysis of the baroreflex curves relating increments in blood pressure to the associated heart rate responses revealed a significantly (p < 0.05) smaller baroreflex sensitivity in female compared with male rats (-1.22 +/- 0.07 and -1.85 +/- 0.15 beats.min-1.mmHg-1, respectively), suggesting an attenuated baroreflex function in females. In age-matched ovariectomized rats, baroreflex sensitivity showed further reduction (-0.93 +/- 0.02 beats.min-1.mmHg-1). Treatment of ovariectomized rats with 17 beta-estradiol significantly (p < 0.05) enhanced the baroreflex sensitivity (-1.41 +/- 0.16 beats.min-1.mmHg-1) to a level that was slightly higher than that of sham-operated female rats. Furthermore, baroreflex sensitivity of ovariectomized estradiol-treated rats was not significantly different from that of age-matched male rats. The vehicle, on the other hand, had no effect on baroreflex sensitivity of ovariectomized rats. These data support our earlier findings that sexual dimorphism exists in baroreflex control of heart rate. More importantly, the present study provides experimental evidence that suggests a facilitatory role for estrogen in the modulation of baroreflex function.

In the present study, the induction of the phosphoinositide signal transduction pathway by 17 beta-oestradiol has been demonstrated in rat vaginal epithelial cells (VEC). We have shown an increase in the metabolism of phosphoinositol lipids by 3H-myoinositol incorporation as well as production of inositol phosphate in VEC in vivo and in vitro following oestradiol administration. Concomitant changes in intracellular calcium levels have also been observed under the influence of oestradiol. To rule out the effects of cytokines, parallel studies were performed using primary cultures of VEC. Oestradiol-induced calcium uptake was seen even in the presence of actinomycin D and cycloheximide which inhibit transcription and translation respectively. Calcium uptake was blocked by neomycin, an inhibitor of phosphoinositol lipid metabolism, and by the oestrogen receptor antagonist tamoxifen. Results suggest that oestradiol induces second messenger pathways in the VEC through specific receptors. Implications of these observations in cellular differentiation processes are discussed.

Cyclic ADP ribose (cADPR) has been shown to trigger Ca2+ release from intracellular stores through ryanodine receptor/channel. In our previous study we observed that all-trans-retinoic acid stimulates cADPR synthesis by ADP ribose cyclase (ADPR cyclase) in cultured epithelial cells. We have now investigated whether cADPR may play a signaling role in action of beta-estradiol (E2), an archetypal steroid superfamily hormone, upon its major target organ, uterus, in vivo. Administration of E2 to gonadectomized rats (0.2 mg/kg per day for 7 days) resulted in an approximately Delta + 300% increase of ADPR cyclase activity in extracts from uterus, but in liver, brain, or skeletal muscle ADPR cyclase was unchanged. Most of the E2-stimulated uterine ADPR cyclase was associated with membranes. The higher ADPR cyclase activity in response to E2 was due to the increase of VMAX without change in Km. Simultaneous administration of estrogen antagonist tamoxifen (8 mg/kg per day) with E2 (0.2 mg/kg per day) prevented an increase in ADPR cyclase. In uterine extracts from E2-treated rats, the rate of cADPR inactivation by cADPR hydrolase and the activity of NADase was increased, but to a much lesser degree than activity of ADPR cyclase. Our results indicate that E2, via action to its nuclear receptors in vivo, increases ADPR cyclase activity in uterus. We propose that some of the estrogen effects, and by extension the effects of other steroid superfamily hormones, upon specialized cellular functions and upon hormone-induced gene expression in target cells, are mediated by cADPR-Ca2+ release pathway.

The mRNA levels of LIV-1 and pS2, two estrogen-responsive genes, are increased by the agents, cholera toxin (CT) plus 3-isobutyl-l-methylxanthine (IBMX), which cause an increase in cAMP in MCF-7 human breast cancer cells. The simultaneous addition of estradiol and CT/IBMX results in a synergistic induction of the two mRNAs. The changes in mRNA reflect changes in transcription of the two genes. Interestingly, the addition of CT/IBMX to estradiol not only causes a greater increase in transcription rate but the increase is longer-lasting that seen with the hormone alone. Stimulation of mRNA levels by CT/IBMX, but not by estradiol, was prevented by cycloheximide. Stimulation by both estradiol and by CT/IBMX was prevented by the antiestrogen, ICI 164387. Transcription of LIV-1 and pS2 genes is by both estradiol and cAMP, via separate mechanisms both requiring the estrogen receptor.

The orphan nuclear receptor steroidogenic factor-1 (SF-1) regulates the expression of several genes involved in the reproductive function and development of the adrenal, the gonads, and the pituitary gonadotropes. It also confers the gonadotrope-specific expression of the glycoprotein hormone a subunit gene by the binding to a gonadotrope-specific element (GSE). In this study, we have shown that SF-1 transactivates the salmon gonadotropin II beta subunit (sGTHII beta) gene expression. SF-1 alone offered a slight but significant enhancement on sGTHII beta promoter activity (7.2 +/- 0.6 fold). However, it stimulated sGTHII beta gene expression dramatically (127 +/- 37 fold) when combined with the estrogen receptor (ER). This synergistic interaction was specific for sGTHII beta promoter as well as for both SF-1 and ER and was estradiol-dose dependent. 5'-Deletion studies of the sGTHII beta promoter identified two putative SF-1 binding sites (GSE) and one previously identified proximal estrogen-responsive element (pERE) at -274 bp involved in this activation. The two GSE sequences located at -354 bp (sGSE(3) and -162 bp (sGSE(2) upstream of the transcription site, although imperfect as compared with the consensus GSE, bound specifically to the in vitro-translated mouse SF-1 protein. 5'-Deletion studies, competition experiments, and site-directed mutagenesis showed that binding to pERE and GSE(2) were necessary for the SF-1/ER synergistic effect. These studies suggest that the synergistic interaction of SF-1 and ER, possibly through cooperative binding or protein-protein interaction, is essential in conferring a cell type-specific expression of the GTHII beta subunit gene.

HMG-CoA reductase inhibitors, such as Lovastatin and Simvastatin, cause cell cycle arrest by interfering with the mitogenic activity of mitogens present in culture media. Cells are induced to pause in G1 and can readily resume growth upon removal of the enzymatic block. Estrogens, acting via their nuclear receptor, are mitogens for different normal and transformed cell types, where they foster cell cycle progression and cell division. In estrogen-responsive MCF-7 human breast cancer cells, but not in non responsive cells, 17 beta-estradiol (E2) induces cells arrested with Lovastatin or Simvastatin to proliferate in the presence of inhibitor, without restoring HMG-CoA reductase activity or affecting the protein prenylation pattern. Mitogenic stimulation of G1-arrested MCF-7 cells with E2 includes primary transcriptional activation of c-fos, accompanied by transient binding in vivo of the estrogen receptor and/or other factors to the ERE and the estrogen-responsive DNA region of this proto-oncogene, as detected by dimethylsulphate genomic footprinting analysis. Mitogenic stimulation of growth-arrested MCF-7 cells by E2 occurs, under these conditions, without evident activation of ERK-1 and -2 kinases, and thus independently from the mitogen-responsive signal transduction pathways that converge on these enzymes.

We report that growth of LNCaP human prostate cancer cells is significantly stimulated (up to 120% above control) by physiological estradiol (E2) concentrations. This growth increase appears to be comparable to that induced by either testosterone or dihydrotestosterone, as also reported by others. This paper presents novel illustrative evidence for estrogen-binding proteins and messenger RNA transcripts in LNCaP cells. In fact, 1) the reverse transcriptase-polymerase chain reaction system documented normal messenger RNA for estrogen receptors (ER); 2) the radioligand binding assay allowed the detection of high affinity, reduced capacity binding sites in both soluble and nuclear cell fractions; and 3) the immunocytochemical analysis showed a consistently intensive staining for both ER and progesterone receptors. Compared to other human estrogen-responsive mammary cancer cells, MCF7 and ZR75-1, ER expression in LNCaP cells was not significantly lower, as shown by levels of the ER transcripts, number of sites per cell, or femtomoles per mg DNA as well as the percentage and intensity of immunocytochemical staining. A relative estimate of ER expression obtained by matching LNCaP with another human prostate cancer cell line, PC3, always displayed significantly and consistently higher levels in LNCaP cells. The detection of relatively high type I ER content in either cell compartment of LNCaP cells was paralleled by a highly intensive staining for progesterone receptors. In addition, evidence that the synthetic androgen R1881 did not compete for type I binding of E2 and that any E2-induced growth was completely reversed by the pure antiestrogen ICI-182,780, but unaffected by the antiandrogen Casodex, clearly suggests that the biological response of LNCaP cells to E2 is mediated via its own receptor.

This study examined endocrine components of sexual orientation of male sheep. Sexual orientation of adult rams was identified through standardized sexual performance tests. Four rams that copulated with ewes, four rams that never mounted females and copulated with males, and eight ewes were used in the experiments. Exogenous estradiol benzoate (50 micrograms, i.m.) stimulated (P < .05) a preovulatory-like LH surge 16-22 hr after administration to females. Estradiol did not (P > .05) affect LH release of heterosexual or homosexual rams. Thirty days after the estradiol challenge, sheep were euthanized and areas of the amygdala (AMY), hypothalamus (HYP), anterior pituitary (AP), and preoptic area (POA) of the hypothalamus were collected. Occupied and unoccupied content of estradiol receptors (ER) was determined. The content of ER in the amygdala of both homosexual rams and ewes was similar, but less than (P < .05) the content of ER in heterosexual rams. The ER content measured in other brain regions did not differ by sex or orientation. In summary, results from these data show that the preovulatory LH surge mechanism that is a characteristic of the female does not occur in either homosexual or heterosexual rams. Conversely, the ER content of the AMY of homosexual rams is similar to that of ewes and differs from the heterosexual male. Differences in ER content between heterosexual and homosexual rams imply that the amygdala serves as a link for input from potential mates. These data suggest that the amygdala not only plays a role in sexual behavior but may be involved in sexual orientation of rams.

Vanadate stimulates growth of the estradiol-responsive MCF-7 cells in the absence of estrogens through a mechanism requiring tyrosine kinase activity. The proliferative effect of vanadate is mediated by estradiol receptor, and is inhibited by three antiestrogens, hydroxytamoxifen, ICI 164,384, and ICI 182,780. Estradiol abolishes the inhibitory effect of ICI 164,384 or ICI 182,780. Before stimulating cell proliferation, vanadate induces accumulation of tyrosine phosphorylation in several proteins including estradiol receptor and epidermal growth factor receptor. In addition, vanadate increases the binding activity of the estradiol receptor for its ligand. This is the first evidence of in vivo association between estradiol receptor tyrosine phosphorylation and its hormone-binding activation. Antiestrogens abolish the vanadate effect on estradiol receptor and epidermal growth factor receptor phosphorylation and reduce it on general protein tyrosine phosphorylation. These findings show that vanadate, apparently through estradiol receptor tyrosine phosphorylation, triggers activity of this receptor, which in turn stimulates protein tyrosine phosphorylation and induces cell proliferation.

Estrogen exhibits a variety of actions, including immuno-modulatory effects, in vivo and in vitro. The mechanism by which estrogen exerts its anti-inflammatory effect is not yet understood. We investigated the possible mechanisms of estradiol acting via the polymorphonuclear leukocytes (PMNs), which are important in the immune response. The agent, 17 beta-estradiol, but not 17 alpha-estradiol, significantly reduced PMNs chemotaxis to FMLP in a dose-dependent manner (control vs estrogen 10(-10)-(-6) M, P < 0.05). Physiological concentrations of estradiol significantly reduced the chemotaxis of PMNs (10(-10) mol). Pre-incubation with clomiphene or tamoxifen which are estrogen receptor antagonists, eliminated the inhibitory effect of 17 beta-estradiol on the chemotaxis of PMNs, restoring it to the control level. These observations suggest that 17 beta-estradiol suppressed the chemotaxis of PMNs by a receptor-dependent mechanism. In addition, the level of estradiol in human plasma, which PMNs were drawn, showed a close, inverse correlation with the PMNs chemotaxis to FMLP (r = -0.821 p < 0.001). Estrogen may modify the activity of neutrophils during the normal menstrual cycle, not only during pregnancy, and influence inflammation.

To better define the mechanisms by which zinc (Zn) deficiency influences periconceptional development, we examined the effects of this developmental insult on uterine estrogen metabolism. CD-1 mice were assigned to 1 of 3 groups (Low Zn, LZ; Control, C; or Replete, R) and fed either a low Zn (< or = 0.3 microgram Zn/g) or control diet (47 micrograms Zn/g) 5 days prior to gestation day (GD) 0 and continuing up to GD 4 during early pregnancy. Mice in the R group were fed the low Zn diet until GD 1 after which they were fed the control diet. Uterine 3H-estradiol binding in vivo was measured on GD 2, GD 3, and GD 4. Binding was similar among groups on GD 2 and GD 3, but was lower on GD 4 in LZ mice than in C and R mice (61% of control value). On GD 4, uterine 3H-estradiol binding in vitro was measured and was lower in LZ mice than in C and R mice (63-74% of control values); the reduction in binding was due to lower receptor number. Thus, Zn deficiency can result in a reduction in uterine estradiol receptors and estradiol binding.

Estradiol increases the number of GnRH receptors in the ewe. Although results from studies conducted in vitro indicate that progesterone may have a negative influence on the number of ovine GnRH receptors, this effect of progesterone has not been documented in vivo. To explore the regulation of GnRH receptors at the level of gene expression, a partial complementary DNA (cDNA) encoding ovine GnRH receptor was isolated using reverse transcription and polymerase chain reaction methodology. This partial cDNA (701 basepairs) was used to isolate a full-length cDNA encoding GnRH receptor from an ovine pituitary cDNA library. Northern blot analysis of RNA from ovine pituitary glands using the partial cDNA as a molecular probe revealed four messenger RNA (mRNA) transcripts at 5.6, 3.8, 2.1, and 1.3 kilobases. In some samples, a fifth transcript at 0.8 kilobases was also evident. GnRH receptor mRNA was not detected in ovine brain, heart, kidney, adrenal, or liver tissues. To examine the regulation of GnRH receptor mRNA and GnRH receptors during the early preovulatory period, relationships among steady state concentrations of GnRH receptor mRNA, numbers of GnRH receptors, and circulating concentrations of progesterone and estradiol during luteolysis were characterized. We hypothesized that during luteolysis, decreased concentrations of progesterone would be associated with increased concentrations of GnRH receptor mRNA and increased numbers of GnRH receptors. On day 11 or 12 of the estrous cycle, luteolysis was induced in 14 ewes by treatment with prostaglandin F2 alpha (PGF2 alpha). Four ewes were treated with saline (saline controls). Anterior pituitary tissue was collected 4 h (n = 4), 12 h (n = 5), and 24 h (n = 5) after treatment with PGF2 alpha or 24 h after treatment with saline and from four untreated ewes on day 11 or 12 of the estrous cycle (untreated controls). Twelve hours after treatment with PGF2 alpha, circulating concentrations of progesterone had decreased (P < 0.05) to 46% of the control values; however, concentrations of estradiol were not different from those in control ewes. Concentrations of GnRH receptor mRNA increased 2-fold during luteolysis and were higher than control values 12 h after PGF2 alpha treatment (P < 0.05). This increase in GnRH receptor mRNA was not accompanied by an increase in the number of GnRH receptors. Twenty-four hours after treatment with PGF2 alpha, concentrations of progesterone in PGF2 alpha-treated ewes had decreased (P < 0.05) to 15% of control values, whereas concentrations of estradiol had increased (P < 0.05) to 321% of control values.(ABSTRACT TRUNCATED AT 400 WORDS)

The participation of adenohypophyseal estradiol receptors in the reinstatement of ovulatory cycles after lactation interruption was investigated. In rats whose pups were removed on day 13 postpartum (LRX), prolactin levels fell as from 1600 h on the same day, estradiol peaked on the morning of day 15 and gonadotropins and prolactin (PRL) surged on the afternoon of day 15. No significant changes in gonadotropins or estradiol levels were observed in rats which remained with their litters (LRP); in these rats daily afternoon surges of PRL were detected. No significant variations in anterior pituitary nuclear or cytosolic estradiol receptors were determined in LRP rats. In the nuclear fraction of LRX rats, an important increase (430.8 +/- 124.9%) in receptor titers was observed on day 15. In these animals a significant increase (34.8 +/- 1.3%) in cytosolic estradiol receptors was observed on day 14, followed by a fall on day 15 (-31.6 +/- 6.6%) in comparison to day 13 levels. The receptor variations observed on day 15 closely resemble estrous cyclic changes determined in adult females. However, an observation which does not resemble those cycle variations is the increase in cytosolic receptors observed on day 14 in LRX rats. This increase may be the consequence of a decrease in dopamine levels induced by pup removal. To our knowledge this is the first time that the involvement of pituitary estradiol receptors in the reinstatement of ovulatory cycles after lactation interruption has been described.

Rams, characterized over a 3-year period as being high (n = 8) or low (n = 10) sexual performers, were euthanized to determine if differences in sexual behavior were associated with differences in hypophyseal and hypothalamic receptors for estradiol. Rams were exposed to estrous ewes and courtship behavior was recorded for 5 hr prior to tissue collection. Blood samples for determination of serum concentrations of luteinizing hormone (LH) were collected every 15 min during the behavior testing period. Hourly blood samples were analyzed for serum concentrations of estradiol (E) and testosterone (T). Rams characterized as high (HP) or low (LP) sexual performers had similar LH profiles; there were no differences noted in basal concentration, number of pulses, or pulse amplitude of LH. There were no differences in serum concentrations of T or E during the behavior testing period between HP and LP rams. Unoccupied receptors for E (moles E bound x 10(-15)) in the stalk-median eminence (SME) (P < 0.05) in HP rams (15.1 +/- 3.1) were greater than those in LP rams (4.6 +/- 1.9). Occupied receptors for E (moles E bound x 10(-15)) in the preoptic area (POA) (P < 0.1) in HP rams (6.0 +/- 0.8) tended to be greater than those in the LP rams (3.8 +/- 0.8). The proportion of total E receptors (occupied+unoccupied) in the POA that were occupied was greater (P < 0.05; 0.41 +/- 0.03 vs 0.23 +/- 0.04) for HP than for LP rams. In contrast, HP rams had less (P < 0.05) occupied E receptors (moles E bound x 10(-15)) in the anterior pituitary gland (AP) than LP rams (152.0 +/- 38.5 vs 337.3 +/- 51.7). The proportion (%) of E receptors that were occupied in the AP in HP rams also tended to be less (P < 0.06) than that in LP rams (8 +/- 2 vs 14 +/- 1). No differences were detected in occupied or unoccupied E receptors in the medial basal hypothalamus, medial amygdala, or stalk-median eminence. In conclusion, differences in occupied receptors for E in the POA and the AP, and unoccupied receptors for E in the SME, were associated with differences in ram sexual behavior.

In order to investigate the possible role of estrogen in the development of cartilage and bone we studied by immunofluorescence immunohistochemistry and autoradiography 26 human embryos and fetuses 7-22 weeks in gestational age associated with pregnancy interrupted for non-medical reasons. In order to demonstrate the presence of estrogen receptors (ERs) in human fetal cartilage, cryostat sections of long bones and lumbar and thoracic vertebrae were prepared for (1) fluorescent immunocytochemistry using an antiidiotypic monoclonal antibody to anti-estradiol receptor monoclonal Ab labeled with fluorescein isothiocyanate (FITC), (2) immunohistochemistry using monoclonal antihuman estradiol receptor antibody, labeled with strept. A-B immunoperoxidase, and (3) autoradiographic localization of estradiol using labeled (3H) 17 beta estradiol. In fetuses aged 10 weeks or older, intranuclear and perinuclear localization of ER was demonstrated by all methods, mainly amongst chondrocytes of the proliferating and higher hypertrophic zones of the epiphyses and in the cartilage of vertebral bodies. These data suggest that estrogen acts directly on chondrocytes of human fetuses through an ER-mediated mechanism.

Endometrial carcinoma is the most common gynaecological malignancy. Progestins are widely used in the treatment of advanced and metastatic disease with about 20-40% response rate. Attempts to develop a predictive test for progestin-sensitivity of endometrial cancers are plagued by the problems of steroid receptor instability and the heterogeneous distribution of progesterone receptor. Successful development of a nude mouse model for human endometrial carcinoma has permitted a detailed investigation of the biological behaviour, hormonal modulation and resistance to progestin therapy. Use of this preclinical model should enhance our understanding of the hormonal mechanisms and lead to improved rational treatment strategies for this malignancy.

Epidermal growth factor (EGF) receptor is inversely related to expression of estrogen receptor (ER) and progesterone receptor in primary breast tumors and is a negative predictor for response to endocrine therapy. To investigate a possible causal role of EGF receptor expression in breast cancer progression to hormone independence, we have created an experimental cell system. Epidermal growth factor receptor complementary DNA was introduced in estrogen-dependent ZR-75-1 breast cancer cells, and the resulting ZR/HERc cells exhibited a mitogenic response to epidermal growth factor, thus bypassing estrogen dependence. This EGF-induced proliferation could not be inhibited by antiestrogens. In addition, we noted changes in cell morphology and keratin expression of EGF-stimulated ZR/HERc cells, suggestive of an altered differentiation state. Furthermore, intolerance of functional ER and EGF receptor signal transduction pathways in ZR/HERc cells was observed during simultaneous activation, which possibly explains the inverse relationship of ER and EGF receptor expression in primary tumors. In contrast to the parental cells, ZR/HERc cells rapidly progressed to a stable ER-negative phenotype when cultured in the presence of the antiestrogen hydroxy-tamoxifen. These results suggest a possible role for EGF receptor in progression of breast cancer to hormone independence.

Selective secretion of FSH, involving no apparent changes in LH secretion, was studied in cycling rat females in the late proestrus and early estrus stages. The preovulatory wave of LH and FSH secretion, observed in the second half of the day of proestrus was associated with elevation of the levels of nuclear estrogenic and androgenic receptors. High concentrations of both receptor types were seen during a secondary elevation of FSH secretion early in the morning in the estrus stage. Administration of phentolamine (alpha-adrenoreceptor blocker) did not influence blood levels of FSH and LH in the estrus stage early hours. Administration of a dopamine blocker haloperidol inhibited the second wave of FSH secretion, this being paralleled by a reduction of the number of estradiol nuclear receptors. The authors suggest that estrogenic and androgenic receptors of adenohypophysis and dopaminergic systems of the brain contribute to the mechanism of regulation of the second phase of FSH increased secretion observed in the early morning hours of the estrus stage, the dopamine effect on FSH release being mediated via estradiol receptors.

The effects of intracranial implants of estradiol in the ventromedial hypothalamus (VMH), the anterior hypothalamus (AH), or the medial amygdala (AMG) on aggression, sexual behavior, and serum estradiol were examined in female Syrian hamsters. Estradiol implants in the VMH, followed by systemic progesterone, stimulated sexual behavior and inhibited aggression. Estradiol implants in other intracranial sites activated sexual behavior but did not reliably inhibit aggression. Intracranially implanted and systemically treated animals had equivalent peripheral estradiol concentrations at sacrifice. These results suggest that: (a) the VMH is an important neural site for estradiol actions on sexual and aggressive behavior, (b) the caudal AH and AMG also may be sites of estradiol action on sexual behavior, and (c) these intracranial implants may only be effective given systemic estradiol exposure or the concurrent stimulation of multiple brain areas.

The hormonal control of ultrasonic vocal behavior in the male Mongolian gerbil was examined by comparing the behavioral effects of androgen with those of estrogen administered to the preoptic-anterior hypothalamic area (POA-AH) in castrates. By measuring radioactivity released from solid "floating" POA-AH microimplants (mean diameter, 141 microns) of testosterone (3H-T, mean weight, 880 ng) in Experiment 1, we found that the steroid had a concentration gradient which fell rapidly from the edge of the microimplant, suggesting restricted diffusion. Using floating microimplants in Experiment 2, we studied the effects of testosterone propionate (TP, 650 ng), estradiol-17 beta benzoate (EB, 439 ng), or cholesterol (C, 478 ng) on rates of a frequency modulated ultrasonic vocalization emitted during sexual interactions. The effects on the upsweep call were compared with those on sexual mounting. The upsweep rate remained significantly below precastration levels in C implanted males. EB reinstated upsweep calling within 5 days, 3 days earlier than TP microimplants. Mounting in EB implanted males was maintained at precastration levels, whereas TP implantation restored mounting to precastration levels only after 5 days. EB was effective in inducing ultrasonic vocalizations when placed in, or near, the sexually dimorphic area (SDA) in the medial preoptic area (POM). Our results indicate that brain mechanisms underlying both ultrasonic vocalizations and mounting are directly sensitive to estradiol (E2) in the male gerbil. We conclude that E2 affects mechanisms in the SDA associated with ultrasonic calling and suggest that T is likely to act via aromatization products in the brain.

We first confirmed the presence of estradiol receptors in the cytosol of rat hepatic sinusoidal endothelial cells and then studied the effects of Sho-saiko-to (TJ-9) on the level of these cytosol estradiol receptors. As a result, we found that estradiol receptors are present in the cytosol of hepatic sinusoidal endothelial cells from rats. Moreover, when these cells were incubated with TJ-9, the level of cytosol estradiol receptors increased. These results suggested that TJ-9 acts on hepatic sinusoidal endothelial cells to increase the level of estradiol receptors, thereby affecting the immune reactions in the liver.

In order to study ultrastructure alterations in prolactin (PRL)-secreting cells, PRL cells of the anterior pituitary glands of partially hepatectomized female rats were observed by the protein A-gold procedure with the electron microscope. Simultaneously, their serum PRL and estradiol (E2) levels were measured by radioimmunoassay. After about 70% hepatectomy PRL cells were remarkably changed, that is active granule extrusion, prominent Golgi vesicles and a hypertrophic rough endoplasmic reticulum were observed. These changes were most remarkable on day 3 after the operation. Significant increases in serum PRL and E2 were also seen following partial hepatectomy. It may be assumed that the morphological changes in PRL cells and the elevation of serum PRL were probably due to surgical stress and to the diminution of E2 receptors in the liver after partial hepatectomy in the rat.

1. The purpose of the present investigation was to study the effect of beta-estradiol crystals implanted in the corticomedial area of the amygdaloid body on the sexual behavior of castrated male rats. 2. The animals were divided into the following groups: group I (N = 9), castrated rats; group II (N = 4), rats which had been castrated and stereotaxically implanted with cholesterol, both groups being used as controls; group III (N = 6), castrated rats with estradiol implants. Latency to the first anogenital exploration, latency to the first mount and mount frequency were determined during the pre-castration and post-castration phases and after the material had been implanted in groups II and III in 10-min observation sessions. 3. There was diminished sexual behavior of the animals in group I without spontaneous recurrence within the period observed. Group II animals, who had undergone implantation of cholesterol, an inert substance, maintained low levels of sexual behavior (post-castration 0.8 +/- 0.7 vs 0.0 +/- 0.0 and 0.5 +/- 0.5 on the 6th and 9th day after implantation, respectively). Group III animals presented a gradual increase in the number of mounts (from post-castration 1.2 +/- 0.5 to 6.5 +/- 2.7 and 4.1 +/- 1.0 on the 6th and 9th day after implantation, respectively) and a decrease of mount latency (from post-castration 431.2 +/- 55.9 to 226.1 +/- 119.6 and 51.0 +/- 28.9 on the 6th and 9th day after implantation, respectively) reaching pre-castration levels on the 6th and 9th day after beta-estradiol implantation.(ABSTRACT TRUNCATED AT 250 WORDS)

Estradiol receptors are discovered in nuclei, cytoplasm and plasmatic membranes of the target cells. Estradiol activates the transmembrane polyphosphoinositide system: it stimulates the protein kinase C translocation from cytosol to cell membranes, the membrane protein phosphorylation being elevated. Transmembrane adenylate cyclase system is also activated. The cAMP system stimulation by estradiol is mediated by protein kinase C, phosphorylating a protein of adenylate cyclase complex. Estradiol causes protein kinases A translocation into the cell nuclei and enhances the protein kinase NII activity. The role of protein phosphorylation, activated by steroid hormones, in the transcription and protein synthesis regulation, is discussed.

We compared the morphology, clonogenic ability, Percoll gradient distribution, estrogen receptor proteins, and interactions with mesenchymal cells in MCF-7 breast tumor cells grown in medium containing fetal calf serum and insulin (FCS-I) or in a defined medium with insulin (ID) as the only growth factor. In the absence of serum and at densities below 5000-8000 cells/cm2, MCF-7 cells required epidermal growth factor, insulin, and thrombin. When cells reached a density of 23,000-26,000 cells/cm2, only insulin was necessary for optimal growth. In ID medium cells showed an enlarged Golgi apparatus and marked plasma membrane modifications, suggesting increased secretory activity. Moreover there was an increase in the release of protein products to the culture medium and a time-dependent ability of these cells to form macrocolonies in soft agar. On the contrary, cells in FCS-I showed no Golgi complex and few plasma membrane modifications. In both culture media tight junctions, desmosomes, and tonofilaments were present. We investigated the effect of conditioned media from MCF-7 cells growing in FCS-I or ID on the growth of primary rat vaginal fibroblasts. The growth of these mesenchymal cells was stimulated by FCS-I medium and inhibited by ID medium. By contrast, the embryonic fibroblast (preadipocyte) line CHEF/18 was also stimulated by FCS-I for the first 48 h, but thereafter ceased growth and acquired lipid droplets and a differentiated morphology. With ID medium, CHEF/18 cells were only partially inhibited with no changes in morphology. The Percoll gradient profiles of ID cells showed the same six fractions of increasing density as recently described. However, there was a progressive increase in subpopulations with higher growth rates and a decrease in the relative amount of the most differentiated cells. A unique feature of the growth analysis of MCF-7 cells in the absence of serum is the increased expression of the estradiol receptor gene. These studies show that the growth and differentiated properties of tumor cells can depend upon the cellular environment and offer a model system in which to further study this modulation.

In leiomyoma and normal myometrium estrogen receptors act independently at low or high levels of the normal serum steroid range in the menstrual cycle. It might be an inherent characteristic of leiomyomas, which results in their progressive growth in the absence of any abnormal stimulation. In the secretory phase of the menstrual cycle, serum progesterone suppresses estrogen receptor concentrations in leiomyoma. In the present study serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) showed direct as well as inverse correlations with estrogen and progesterone receptors in different phases of the menstrual cycle.

Oestradiol exerts an important modulatory influence on the release of prolactin which is accomplished partly through disruption of the inhibitory influence of dopamine. We have focused on the status of the anterior pituitary D2 dopamine receptor in female rats treated chronically with oestradiol or progesterone. A direct membrane effect of these steroids on the dopamine system was also investigated in vitro. Both steroids affected the status of the D2 receptor, oestradiol decreasing the number of sites in vitro and progesterone increasing it both in vitro and in vivo. The in vitro studies demonstrated that these steroids exert a direct membrane effect on the D2 receptor. These results correlated with an in vitro short-term physiological effect of oestradiol and progesterone on the dopaminergic inhibition of prolactin release, oestradiol decreasing it while progesterone had the opposite effect. Binding studies with [3H] oestradiol on pituitary membranes revealed a site for oestradiol of high affinity and low capacity, indicating that oestradiol's membrane effects could be mediated by a specific receptor. In vivo treatment with oestradiol also induces proliferation of prolactin-secreting cells (lactotrophs). We focused on the effect of oestradiol on protein kinase C activity, which is involved in both secretion and proliferation. In female rats treated with oestradiol total protein kinase C activity was increased by 74% (particulate 90%, soluble 71%) in comparison with controls. This effect was reversed by concomitant treatment with a dopamine agonist. Thus in the pituitary oestradiol and progesterone affect the characteristics of membrane components that are implicated in the physiological control of the cell. Whether these effects are post-transcriptional only or are also mediated through direct membrane mechanisms needs further investigation.

Stress-related activation of the hypothalamic-pituitary-adrenal axis (HPA) is associated with suppression of the reproductive axis. This effect has been explained by findings indicating that corticotropin-releasing hormone suppresses hypothalamic gonadotropin-releasing hormone (GnRH) secretion via an opioid peptide-mediated mechanism, and that glucocorticoids suppress both GnRH and gonadotropin secretion and inhibit testosterone and estradiol production by the testis and ovary, respectively. To evaluate whether glucocorticoids suppress the effects of estradiol on its target tissues, we examined the ability of dexamethasone to inhibit estradiol-stimulated uterine and thymic growth in ovariectomized rats. Estradiol alone, given daily for 5 days, caused dose-dependent uterine and thymic growth. Dexamethasone alone, given daily for 5 days, caused a dose-dependent decrease in body weight gain and in thymic growth. When estradiol and dexamethasone were administered simultaneously, however, body weight gain and thymic growth were also inhibited (p less than 0.05). Dexamethasone decreased estradiol-induced uterine cytosolic and nuclear estrogen receptor concentrations (E2 R0, p less than 0.05; E2nR0, respectively), but had no effect on estradiol-induced progesterone receptor concentrations (P4R0, p greater than 0.05). Levels of uterine glucocorticoid receptors were not affected by estrogen and/or dexamethasone treatment. These findings suggest that stress levels of glucocorticoids, administered over a 5-day interval, block the estradiol-stimulated growth of female sex hormone target tissues. This effect may be partially mediated by a glucocorticoid-induced decrease of the estradiol receptor concentration. Thus, another mechanism by which the HPA may influence reproductive function during stress is by a direct effect of glucocorticoids on the target tissues of sex steroids.

The synergistic cytotoxicity of the combined treatment of hyperthermia and estradiol was demonstrated. This effect was measured for both clonogenic efficiency and membrane function, whose function was expressed as the initial rate of thymidine (dThd) incorporation. V-79 cells in a suspension culture were simultaneously treated with 10(-9)-10(-4) mg/ml estradiol at temperatures of 41-43 degrees C in a water bath. The survival rate of the V-79 cells decreased as the temperature increased. However, there was little decrease from treatment by estradiol alone. The survival rate for combined treatment by both heat and estradiol was lower than that of the separate treatment by either hyperthermia or estradiol alone, and the results were statistically significant. The values for Vmax (maximum reaction velocity) of hyperthermia decreased as the temperature increased, although the Vmax value of estradiol treatment alone changed little. The 1/Vmax value for the combined treatment of heat and estradiol increased in comparison with that of heat treatment alone. However, the Km (Michaelis constant) value showed little correlation with the survival rate. Based on these results, we propose that estradiol enhances cell membrane damage by hyperthermia.

The stimulation of yolk protein synthesis by estrogen is a characteristic of female non-mammalian vertebrates; in mammals, however, vitellogenesis has been suppressed as a corollary of the evolution of viviparity. It is our hypothesis that progesterone has a dual role in this phylogenetic trend; a) to inhibit myometrial contraction and thus set the stage for internal development of embryos and associated placentation, b) to inhibit yolk protein synthesis as placentation became an efficient direct supply of nutrients to the fetus. We have presented evidence that in the reptiles, the central vertebrate group from which the ancestors of modern mammals evolved, the control of yolk protein synthesis is complex, involving both pituitary and ovarian steroids (estradiol, testosterone and progesterone). This system provides an excellent model for the multihormonal contents of gene regulation involving both + and - controls.