Single Injection Of Estradiol Research Articles

Modulatory effects of 17β-estradiol on acute lung inflammation after total occlusion of the descending aorta in male rats

As a consequence of systemic inflammation caused by ischemia and reperfusion (I/R) due to aortic occlusion, the lungs can exhibit increased microvascular permeability, local release of pro-inflammatory mediators, and leukocyte infiltration. Lung tissue infiltration by activated neutrophils is followed by acute respiratory distress syndrome, which is linked to acute pulmonary microvascular damage, high mortality rates, and organ dysfunction. Previous studies have demonstrated that female sex hormones modulate the inflammatory response and that prophylactic treatment with 17β-estradiol (E2) can prevent fatalities and preserve mesenteric perfusion and intestinal integrity after ischemia/reperfusion induced by aortic occlusion. In this study, we focused on the protective effects of estradiol after aortic ischemia/reperfusion by evaluating lung injury and endothelial alterations. Upon anesthesia and mechanical ventilation, male rats were subjected to aortic occlusion for 20 min, followed by 2 h of reperfusion. In parallel, one group of rats received a single injection of estradiol (280 µg/kg, i.v.) 30 min before ischemia. We observed increased serum concentrations of IL-1β, IL-6 and IL-10 in the I/R rats and E2 was able to reduce them. E2 effects after 2 h of reperfusion resulted mainly in decreasing of edema, iNOS expression and preventing leukocyte infiltration. Overall, our data indicate that estradiol might be a supplementary approach to deal with systemic processes and lung deterioration.

Sex differences and sex hormones in anxiety-like behavior of aging rats

Sex differences in the prevalence of affective disorders might be attributable to different sex hormone milieu. The effects of short-term sex hormone deficiency on behavior, especially on anxiety have been studied in numerous animal experiments, mainly on young adult rats and mice. However, sex differences in aged animals and the effects of long-term hypogonadism are understudied. The aim of our study was to analyze sex differences in anxiety-like behavior in aged rats and to prove whether they can be attributed to endogenous sex hormone production in males. A battery of tests was performed to assess anxiety-like behavior in aged female, male and gonadectomized male rats castrated before puberty. In addition, the aged gonadectomized male rats were treated with a single injection of estradiol or testosterone or supplemented with estradiol for two-weeks. Female rats displayed a less anxious behavior than male rats in most of the conducted behavioral tests except the light-dark box. Long-term androgen deficiency decreased the sex difference in anxiety either partially (open field, PhenoTyper cage) or completely (elevated plus maze). Neither single injection of sex hormones, nor two-week supplementation of estradiol in gonadectomized aged male rats significantly affected their anxiety-like behavior in the elevated plus maze. In conclusion, our results confirm sex differences in anxiety in aged rats likely mediated by endogenous testosterone production in males. Whether long-term supplementation with exogenous sex hormones could affect anxiety-like behavior in elderly individuals remains to be elucidated.

Progesterone is primary regulator of Cdk2ap1 gene expression and tissue-specific expression in the uterus

Proliferation of endometrial cells is a prerequisite step for functional differentiation in the uterus. A tumor suppressor gene, Cyclin-dependent kinase 2-associated protein 1 (Cdk2ap1) mRNA was detected in the pregnant uterus and was suggested to be involved in cell proliferation. However, its roles and the mechanisms regulating its expression are largely unknown. In this study, the role of steroid hormones in the expression of Cdk2ap1 was examined using RT-PCR, Northern blotting and in situ hybridization methods. Cdk2ap1 mRNA was highly expressed during the proestrus phase and was mainly localized in the epithelium and subepithelium. Its expression was induced by a single injection of estradiol and progesterone, but the effect of progesterone was stronger than that of estradiol. Injections of progesterone (P1,2) on 2 consecutive days induced Cdk2ap1 expression in the endometrium with the same patterns observed in the proestrus phase, but injections of estradiol (E1,2) on 2 consecutive days did not induce expression. The Cdk2ap1 mRNA level was decreased by combined treatment of progesterone and estradiol (E1+P2,3). RU486 suppressed completely the Cdk2ap1 mRNA expression in P1,2 while ICI 182,780 did not in E1+P2,3. In the uteri on day 4 of gestation, expression of Cdk2ap1 also was regulated by progesterone as expected. Cdk2ap1 mRNA expression was totally suppressed by RU486 but not by ICI 182,780. Thus, it is suggested that Cdk2ap1 expression is primarily regulated by progesterone and the progesterone receptor in uterus and is mainly localized to proliferating tissues.

Aging alters the expression of genes for neuroprotection and synaptic function following acute estradiol treatment

This study used microarray analysis to examine age-related changes in gene expression 6 and 12 h following a single estradiol injection in ovariectomized mice. Estradiol-responsive gene expression at the 6 h time point was reduced in aged (18 months) animals compared with young (4 months) and middle-aged (MA, 12 months) mice. Examination of gene clustering within biological and functional pathways indicated that young and MA mice exhibited increased expression of genes for cellular components of the synapse and decreased expression of genes related to oxidative phosphorylation and mitochondrial dysfunction. At the 12 h time point, estradiol-responsive gene expression increased in aged animals and decreased in young and MA mice compared with the 6 h time point. Gene clustering analysis indicated that aged mice exhibited increased expression of genes for signaling pathways that are rapidly influenced by estradiol. The age differences in gene expression for rapid signaling pathways may relate to disparity in basal pathway activity and estradiol mediated activation of rapid signaling cascades.

Estrogen and Tumors: For Better or for Worse?

In their Report “Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production” (6 July, p. [121][1]), W. E. Naugler et al. clearly demonstrated the role of IL-6 in the development of hepatocellular carcinoma (HCC) in mice. In addition, a single estradiol injection

MATING SHUTS DOWN ESTRADIOL NONGENOMIC SIGNALING PATHWAY IN THE RAT OVIDUCT

A single injection of Estradiol (E2) given before mating accelerates egg transport (ET) in the rat oviduct via non transcriptional pathways involving cAMP, IP3 and protein kinases (PK), but does it via genomic pathways when it is given after mating. This change in signaling pathways induced by mating is referred to as “intracellular pathway shifting” (IPS), and it can be elicited in complete absence of the male, by mechanical stimulation of the genital area or by artificial insemination. In order to characterize IPS, we compared genomic and nongenomic pathways responsive to E2 in mated and nonmated rats. Estradiol increased cAMP level in the oviduct of nonmated rats, but failed to do so after mating. Moreover, the adenylyl cyclase activator, forskolin, accelerated ET only in nonmated rats, suggesting that mating-associated signals shut down the E2 nongenomic pathway up and down stream of cAMP production. The pattern of gene expression induced by E2 in the oviduct was different in mated and nonmated rats. Estradiol increased transcript levels of 40 genes common to both conditions, e.g. progesterone receptor and brain creatine kinase, indicating that a classical genomic pathway responsive to E2 operates both in mated and nonmated rats. Estradiol increased the expression of other 37 genes in nonmated rats that were unresponsive after mating. StAR is one of these and its increased expression was suppressed by ICI 182780 and the adenylyl cyclase inhibitor SQ22536. Only 2 genes, adenosine monophosphate deaminase 3 and calbindin 9kDa, increased their mRNA levels in response to E2 exclusively in mated rats. Local injection of a morpholino antisense oligonucleotide that inhibited the E2-induced increase expression of calbindin 9kDa protein, partially prevented E2 from accelerating ET in mated rats, but had no effect before mating. Microarray analysis of the oviduct of mated and nonmated rats at equivalent times corresponding to 6 h post coitum identified 21 genes whose expression was up or downregulated by mating. The level of transcript coding for Catechol-OMethyltransferase (COMT), which produces methoxyestradiols from hydroxyestradiols, diminished 6 times and this was confirmed by Real Time PCR. O-methylation of 2-hydroxyestradiol was up to four orders of magnitude higher in oviductal protein extracts from nonmated than from mated rats. Local application of a COMT inhibitor (OR 486) to the oviduct prevented E2 from accelerating ET only in nonmated rats, and local application of 2-methoxyestradiol (2ME) mimicked the effect of E2 on ET only in nonmated rats. The effect of 2ME on ET was blocked by local administration of the PK blocker H-89 or by ICI 182780, but not by Actinomycin D or OR486. In conclusion, a nongenomic pathway, activated by E2 metabolites generated by COMT, mediates the effect of E2 on ET and a classical E2 genomic pathway not involved in the kinetic effect of E2 operates simultaneously in the oviduct before mating. Mating-associated signals shut down this E2 nongenomic pathway, up and downstream of 2ME, and up and downstream of cAMP production, allowing increased expression of genes involved in accelerated ET after mating. The specific signal(s) that elicits IPS, the primary event(s) and cell-phenotype(s) involved, and the biological significance of mating-induced IPS remain to be disclosed. FONDECYT 1030315, PROGRESAR PRE 004/03 and a Scholarship from CONICYT to A P-B. (poster)

Sexual Behavior Activity Tracks Rapid Changes in Brain Estrogen Concentrations

Estrogens are classically viewed as hormones that bind to intracellular receptors, which then act as transcription factors to modulate gene expression; however, they also affect many aspects of neuronal functioning by rapid nongenomic actions. Brain estrogen production can be regulated within minutes by changes in aromatase (estrogen synthase) activity as a result of calcium-dependent phosphorylations of the enzyme. To determine the effects of rapid changes in estrogen availability on male copulatory behavior, we mimicked in male mice the rapid upregulation and downregulation of brain estrogen concentration that should occur after inactivation or activation of aromatase activity. A single injection of different aromatase inhibitors [Vorozole, 1,4,6-androstatrien-3,17-dione (ATD), or its metabolite 17-OH-ATD (1,4,6-androstatrien-17beta-ol-3-one)] almost completely suppressed male sexual behavior (mounts and intromissions) expressed 10-20 min later by C57BL/6J mice but did not affect behavior in aromatase knock-out (ArKO) mice, activated by daily injections of estradiol benzoate, thereby confirming the specificity of the behavioral inhibition observed in wild-type mice. The rapid ATD-induced inhibition was reversed by the simultaneous injection of a large dose of estradiol. A single injection of estradiol to ArKO mice also activated male sexual behavior within 15 min. Thus, rapid increases or decreases in brain estrogen concentrations are followed within minutes by corresponding changes in male sexual behavior. Sexual behavior can thus be used to monitor changes in local estrogen concentrations and analyze the mechanisms mediating the rapid decline in estrogen signaling that takes place after inhibition of estrogen synthesis.

Effect of oestradiol and progesterone on the instant and directional velocity of microsphere movements in the rat oviduct: gap junctions mediate the kinetic effect of oestradiol

The oviducal transport of eggs to the uterus normally takes 72-96 h in the rat, but this is reduced to less than 20 h after a single injection of oestradiol (E2). This accelerated transport is associated with an increased frequency of pendular movements in the isthmic segment of the oviduct, with increased levels of the gap junction (GJ) component Connexin (Cx) 43, and is antagonised by progesterone (P). In the present study, we investigated the effect of these hormones on the instant and directional velocity of pendular movements and the role of the GJ and its Cx43 component in the kinetic response of the oviduct to E2 and P. Using microspheres as egg surrogates, microsphere instant velocity (MIV) was measured following treatment with E2, P or P + E2, which accelerate or delay egg transport. Microspheres were delivered into the oviduct of rats on Day 1 of pregnancy and their movement within the isthmic segment was recorded. Oestrogen increased MIV with faster movement towards the uterus. After P or P + E2, MIV was similar to that in the control group. Two GJ uncouplers, namely 18 alpha- and 18 beta-glycyrrhetinic acid, blocked the effect of E2 on MIV. Connexin 43 mRNA levels increased over that seen in control with all treatments. In conclusion, the effects of E2 on MIV resulted in faster movements that produced accelerated egg transport towards the uterus. Gap junctions are probably involved as smooth muscle synchronisers in this kinetic effect of E2, but the opposing effects of E2 and P are not exerted at the level of Cx43 transcription.

Estradiol rapidly activates male sexual behavior and affects brain monoamine levels in the quail brain

Steroids are generally viewed as transcription factors binding to intracellular receptors and activating gene transcription. Rapid cellular effects mediated via non-genomic mechanisms have however been identified and one report showed that injections of estradiol rapidly stimulate chemoinvestigation and mounting behavior in castrated male rats. It is not known whether such effects take place in other species and what are the cellular underlying mechanisms. We show here that a single injection of estradiol (500 μg/kg) rapidly and transiently activates copulatory behavior in castrated male quail pre-treated with a dose of testosterone behaviorally ineffective by itself. The maximal behavioral effect was observed after 15 min. In a second experiment, the brain of all subjects was immediately collected after behavioral tests performed 15 min after injection. The preoptic area—hypothalamus (HPOA), hindbrain, telencephalon and cerebellum were isolated and monoamines measured by HPLC-ED. Estradiol increased levels of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) and 5-HIAA/serotonin ratios in the telencephalon and hindbrain independently of whether animals had mated or not. Estradiol also affected these measures in HPOA and cerebellum but this effect was correlated with the level of sexual activity so that significant effects of the treatment only appeared when sexual activity was used as a covariate. Interactions between estradiol effects and sexual activity were also observed for dopamine in the HPOA and for serotonin in the hindbrain and cerebellum. Together, these data demonstrate that a single estradiol injection rapidly activates male sexual behavior in quail and that this behavioral effect is correlated with changes in monoaminergic activity.

Estrogen changes mitosis orientation in the uterine epithelia

Objectives: Estrogens can lead to hyperplasia and cancer formation in the uterus, ovary and mammary gland. However, key factors involved in this process are not defined at present. It is possible that estrogens primarily affect more global tissue-organising parameters as composition of tissues in three-metric space, that is brought about changes in the orientation of the plane of cell division. There are no data about estrogen action on mitosis orientation in the uterus. Study Design: Mitosis orientation, proliferative activity and general histology were examined in the uterus, jejunal and colonic crypts, and epidermis of ovariectomized rats 0, 24, 36 and 48 h after a single injection of estradiol, and in the uterus of ovariectomized mice received injections with estradiol once-a-week for 30, 60 and 90 days. Results: All mitoses in luminal and glandular epithelia of the uterus of ovariectomized rats aligned parallel to the basement membrane, and estradiol treatment leads to appearing of mitoses oriented perpendicular to the basement membrane of the epithelium. Together with increase in the proliferative activity, the number of perpendicular oriented mitoses in uterine epithelia of ovariectomized rats is significantly increased after a single injection of estradiol. During endometrial hyperplasia formation in mice, that is induced by chronic estrogen treatment, the number of perpendicular oriented mitoses in uterine epithelia become much more higher. In jejunal and colonic crypts, epidermis of ovariectomized rats, most of mitoses disposed parallel to the basement membrane and an injection of estradiol did not affect mitosis orientation and activity of proliferation. Conclusions: Estradiol effect on mitosis orientation may be considered as specific. Changes in mitosis orientation are probably responsible for estrogen-dependent hyperplasia and cancer formation in the uterus.

Modulation of rat epididymal?-glutamyl transpeptidase by nonylphenols

1. gamma-Glutamyl-transpeptidase (gamma-GTP), present at low levels in the testis, seminal vesicle, prostate gland and epididymis in rat at 4 days of age, showed rapid developmental increases at the time of weaning. 2. Administration of nonylphenols (NP) to the neonatal male rat pup (from days 1 to 15) impaired the subsequent development of gamma-GTP in the epididymis but not in the testis, seminal vesicles or prostate gland. 3. Single injection of NP to weaned pups at approximately 22 days of age decreased gamma-GTP in the epididymis but not in other male accessory sex organs. This effect was transient, dose-dependent and blocked by the oestrogen receptor-specific antagonist ICI 182,780. 4. Single injection of oestradiol to weaned rat at approximately 22 days of age also decreased gamma-GTP in the epididymis but not in the testis, prostate gland or seminal vesicles. 5. In in vitro assays, NP did not inhibit epididymal gamma-GTP activity even at 100 microM final concentration. Under similar conditions, acivicin, a specific inhibitor for gamma-GTP, showed a dose-dependent inhibition of gamma-GTP activity. 6. It is suggested that NP impair gamma-GTP expression in the epididymis of developing male rat and act in part via the oestrogen receptor.

Role of mast cells in oestradiol effects on the uterus of ovariectomized rats.

The role of mast cells and their main secretory products in the effects of oestradiol on the uterus was investigated. Ovariectomized rats were treated with a single injection of oestradiol (10 micrograms per rat, i.m.) or vehicle together with drugs affecting the activity of mast cells, cromoglycate (10 mg per rat, i.m.), which diminishes the degranulation of mast cells, or compound 48/80 (0.5 mg per rat, i.m.), which enhances this process. Oestradiol or vehicle was also administered with two important secretory products of mast cells, heparin (0.4 mg per rat, i.m.) or histamine (2 mg per rat, i.m.). All drugs were injected simultaneously with oestradiol (first injection) and then every 6 h until the animals were killed. Observations were performed at 24, 36 and 48 h after oestradiol or vehicle injection. The condition of mast cells was determined by the percentage of degranulated mast cells in sections stained with toluidine blue. Oestradiol-induced effects in the uterus were estimated by the mitotic index, proliferating cell nuclear antigen-labelling index, DNA content, volumes of cells, nuclei and nucleoli in the luminal epithelium, glandular epithelium and stroma cells of the endometrium. Cromoglycate treatment resulted in a decrease in both mast cell degranulation and all examined oestradiol effects in the uterus at all periods of observation. Compound 48/80 increased mast cell degranulation and expression of one aspect of oestradiol effects on the volumes of cell compartments. Histamine or heparin led to a marked increase in the cell, nucleus and nucleolus volumes in all uterine structures. However, heparin produced a depression in proliferation, whereas histamine had a weak transient stimulating action on this process. No effects of the protocols were found in the absence of oestradiol treatment. These results suggest that mast cells are involved in the realization of oestrogen action, including the stimulation of cell growth and proliferation in the uterus, and that the effect of mast cells is mediated by both histamine and heparin.

Phorbol ester inhibition of estrogen-induced uterine deoxyribonucleic acid synthesis.

Protein kinase C (PKC) is a key regulatory enzyme in the control of growth and differentiated function in many cell types. Recently it has become clear that cross talk occurs between PKC and steroid hormone-activated signaling pathways. In this work we have thus used the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to investigate the relationship between PKC activation and estrogen-induced proliferation in an in vivo model of hormone action, the immature rat uterus. A single injection of estradiol (E2) to immature female animals increases DNA synthesis in all major uterine cell types. Administration of TPA alone, simultaneous administration of TPA and E2, or administration of TPA 12 h after E2 did not alter uterine DNA synthesis. However, administration of TPA 6 h after E2 markedly decreased [3H]thymidine incorporation and the labeling indices in uterine epithelial, stromal, and myometrial cells. This inhibition represents a decrease in DNA synthesis per se rather than a change in the time course of the tissue response to the hormone. The inhibitory effect of TPA was reversible within 72 h and did not appear to be due to a decrease in the level or degree of occupancy of uterine estrogen receptors. These results suggest that a discrete regulatory event(s) in the pathway of estradiol-induced proliferation is inhibited by PKC activation approximately 6 h after hormonal stimulation.

Oestrogen and progesterone control basic fibroblast growth factor mRNA in the rat uterus.

Cell proliferation and differentiation in the rodent uterus are probably controlled by the interaction of female sex steroids with polypeptide growth factors. Uterine basic fibroblast growth factor (bFGF) mRNA was measured by RNase protection during the time (days 2-4) of endometrial cell proliferation in the pregnant rat. bFGF transcripts were detected at each of the 3 days of pregnancy examined. To investigate the influence of oestrogen and progesterone on bFGF mRNA accumulation, ovariectomized rats were treated with oestradiol for 48 h followed by a single injection of oestradiol, progesterone, the two steroids co-injected or oil vehicle alone. Uterine RNA was collected 6 h after the last hormone injection. Steroid treatments increased steady-state uterine bFGF mRNA compared with vehicle control animals as measured by RNase protection. Northern blot analysis of c-fos and c-jun mRNAs from these same treatment groups revealed increased protooncogene expression in the uterus of hormone treated rats compared with the control animals. Temporal analysis of bFGF mRNA in ovariectomized rats at 1, 3 and 6 h after acute oestrogen and oestrogen-progesterone co-administration showed a dual pattern of transcript accumulation. Both hormone treatments increased bFGF mRNA within 1 h compared with vehicle injected rats. Co-administration of the two hormones, however, repressed bFGF mRNA accumulation relative to oestrogen at 3 and 6 h. Together, these studies provide evidence that bFGF control of uterine cell proliferation in pregnant rats can occur from newly synthesized bFGF. Moreover, the results suggest that progesterone is a potent stimulator of bFGF expression in the uterus.

Lack of gonadotropin-positive feedback in the male rat is associated with lack of estrogen-induced synaptic plasticity in the arcuate nucleus.

We have demonstrated that estrogen induces synaptic reorganization in the hypothalamic arcuate nucleus of female rats during the ovarian cycle and proposed that estrogen-induced synaptic retraction plays a role in the disinhibition of gonadotropin secretion that occurs during the afternoon of proestrus. This so-called positive feedback of gonadotropins is developmentally determined. It is present in female rats and absent in males. To confirm the role of the estrogen-induced synaptic retraction in positive feedback, we tested whether administration of estrogen to male rats also fails to induce synaptic remodeling of the arcuate nucleus. Male and female rats were gonadectomized and studied as follows. One month following gonadectomy, animals received either a single injection of estradiol (100 micrograms/animal in sesame oil; 12 males and 12 females) or vehicle (6 males and 6 females). Twenty-four hours following injections, all animals in the vehicle-injected group and 6 animals of each sex in the treatment groups were sacrificed, while the rest of the animals were killed 48 h following the hormone injections (6 per group). As expected, quantitative electron microscopic analysis of the female arcuate nuclei revealed that compared to oil-injected controls, estradiol induced drastic decreases in the overall synapse counts by 24 h (121 +/- 10 vs. 74 +/- 5 synapses/1,000 microns membrane; p < 0.05). Synaptic counts had recovered to control levels by 48 h. On the contrary, in males, estradiol treatment did not cause changes in the total synapse counts at either time. As a further control, the lack of an estrogen-induced gonadotropin surge in long-term castrate males was also confirmed. Our study confirmed that in males estradiol does not alter the net synaptology of the arcuate nucleus or cause gonadotropin positive feedback. This is in clear contradistinction to females which show both synaptic plasticity and gonadotropin-positive feedback upon receiving exogenous or endogenous estrogen. The lack of estrogen-induced synaptic plasticity may be an underlying mechanism in the abolishment of positive gonadotropin, feedback in developing males and the development of constant estrus in aging female rats.

Rat uterine stromal cells: thrombin receptor and growth stimulation by thrombin.

The estrogen-stimulated maturation of the immature rat uterus is mediated by peptide growth factors whose expression is regulated by estradiol. We present evidence that thrombin is a uterine growth factor. When an immature rat is given a single injection of estradiol, the uterus increases 50% in wet weight within 3 h through the imbibition of water and plasma proteins, including prothrombin. Tissue factor, the initiator of coagulation, is induced 3- to 4-fold over the same time period. Thrombin is generated in situ from prothrombin through the coagulation cascade. It acts as a growth factor through the proteolytically activated thrombin receptor. Thrombin's role as a growth factor in uterine stromal cells is proven by two lines of evidence: demonstrations that the proteolytically activated thrombin receptor is present and that cultured cells are stimulated to grow by thrombin. Thrombin receptor in the uterus is demonstrated by reverse transcription-PCR for receptor messenger RNA by specific [125I]peptide labeling of a membrane-bound binding protein of about 60 kDa and by Western blot with a thrombin receptor antipeptide antibody. Thrombin's effectiveness as a growth factor is shown by thrombin-stimulated growth of primary stromal cell cultures, with maximum stimulation at 100 nM. That the effect is mediated by the proteolytically activated thrombin receptor is shown by the inhibition of growth by hirudin, a highly specific inhibitor of thrombin; the absence of enhanced growth with Pro-Phe-Arg-chloromethyl ketone-thrombin, an active site-inhibited thrombin derivative; and the stimulation of growth by the thrombin receptor-activating peptide.

Identification and characterization of an estrogen-responsive element binding protein repressed by estradiol.

Cytosolic proteins from uteri of 19-day-old rats were analyzed by an electrophoresis mobility shift assay (EMSA) using a 31 base pair DNA probe containing an estrogen-responsive element (ERE) from the vitellogenin A2 gene. EMSA identified three distinct cytosolic protein-DNA complexes that are separable by Q-Sepharose anion exchange chromatography into an estrogen receptor (ER)-containing fraction (150 mM NaCl eluate) and a non-ER-containing fraction (250 mM NaCl eluate). We thus refer to the non-ER fraction as the ERE binding protein (ERE-BP). The ERE-BP-containing fraction was repressed to 40-50% of its normal levels following a single injection of estradiol. In addition, ERE-BP levels were repressed to the same extent (greater than 50%) by day 20 of the rat's gestational period. Examination of the expression pattern of ERE-BP shows that this activity is differentially expressed in both estrogen-responsive and nonresponsive tissues, with the highest levels of expression occurring in the pituitary. We next examined the specificity of ERE-BP binding by competition analysis using DNA sequences corresponding to binding sites of several known transcription factors. ERE-BP was found to be specific for both the ER binding site (ERE) and TATA binding protein binding sites. Furthermore, saturation analysis demonstrated that ERE-BP binds to the ERE and TATA binding protein sequences with an apparent Kd of 1.2 and 0.12 nM, respectively. Partial purification of ERE-BP using three chromatography steps (Q-Sepharose, hydroxyapatite, and Sephacryl S300) followed by sodium dodecyl sulfate analysis indicated the presence of three major protein bands (p102, p81, and p48) as judged by Coomassie staining. UV cross-linking of the ERE-BP/DNA complex followed by sodium dodecyl sulfate analysis-polyacrylamide gel electrophoresis analysis indicates that the 48 kDa band seen in the final, partially purified fraction correlates with the ERE-BP activity. Thus, this study has identified a unique uterine cytosolic protein that binds to the ER binding site and may influence ER binding.

Is estrogen-induced pituitary hyperplasia and hyperprolactinaemia mediated by angiotensin II?

Human and rat anterior pituitary glands (APG) contain local renin-angiotensin systems (RAS). In the rat APG renin, renin mRNA, angiotensin converting enzyme (ACE) and angiotensin II (A II) have been identified within gonadotrophs (Naruse et al. 1986; Deschepper et al. 1986; Thomas and Sernia 1990). Angiotensinogen (AGEN) and AGEN mRNA have also been found in the rat APG but their localization remains still unclear (Ganong and Deschepper 1989; Healy 1992). Main target sites for A II action in APG seem to be lactotrophs. Specific A II binding sites have been identified mainly on lactotrophs and A II has been shown to stimulate prolactin (PRL) secretion (Aguilera et al. 1982; Denef and Schramme 1985). Recently, we have found that A II stimulated the proliferation of lactotrophs isolated from the estrogen-induced pituitary tumors in the rat (Kunert-Radek and Pawlikowski 1992). Moreover, an ACE inhibitor, enalapril, was found to prevent the pituitary proliferative response to a single injection of estradiol in rats (Mucha et al. 1993). Estrogens are well known to stimulate the pituitary cell proliferation and tumorigenesis and the estrogen-induced APG hyperplasia involves mainly lactotrophs (Zondek 1936; Mietkiewski 1959; Lloyd et al. 1975). Estrogens have been also found to enhance the prolactin secretion (Nicoll and Meites 1962). On the other hand, estrogens were found to increase the AGEN plasma levels (Menard et al. 1970) and to enhance AGEN mRNA expression in the liver, brain (Kunapuli et al. 1987) and pituitary (Healy et al. 1992). Thus, a question arises whether A II is involved in the estrogen action on APG. The aim of the experiments presented below was to test the hypothesis in question.

Mouse estrogen receptor gene knock out: Molecular characterization and resulting phenotypes

The estrogen receptor (ER) is thought to play a crucial role in the regulation of many l i f e processes, including development, reproduction and normal physiology. Because there have been no known mutations of the estrogen receptor in normal t issue of humans or animals, i t s presence and t issue d is t r ibu t ion is thought to be essential for surv iva l . Using the techniques of homologous recombination, we have disrupted the ER gene in mouse embryonic stem cel ls and have produced a l ine of transgenic mice possessing the altered ER gene. The mouse ER gene was disrupted by insert ing a 1.8kb PGK-Neomycin sequence exon 2, approximately 280bp downstream of the s tar t codon. The correct target ing of the disruption was demonstrated by both Southern blot analysis and polymerase chain reaction (PCR). Western blot analysis of uterine preparations from females that are homozygous for the ER gene disruption showed no detectable ER protein. Heterozygotes had one hal f the level of ER protein. Treatment with estradiol at 40ug/kg/day for three days resulted in a 3-4 fold increase in uterine wet weight and vaginal corn i f icat ion in the wi ld type females but e l i c i t ed no s imi la r response in the homozygous mutant females. This data was fur ther supported by a 3H-Thymidine incorporation assay on uterine t issue of s im i la r l y treated mice. Lactoferr in, an estrogen responsive gene in the uterus, was also assayed by northern blot . Ovariectomized wi ld type mice treated with a single estradiol in ject ion showed a 350-fold induction in lac to fer r in mRNA, while ovariectomized homozygous ER mutant females showed no detectable response. Both male and female animals survive to adulthood with normal gross external phenotypes. As expected, females are i n f e r t i l e and demonstrate hypoplastic uter i and hyperemic ovaries with no apparent corpora lutea. Males are also i n f e r t i l e , with atrophy of the testes. Preliminary analysis of the mammary glands of ER mutant females at four months of age showed a pr imi t ive ductal rudiment that extended only 5-6mm into the fat pad compared to a f u l l y developed ductal tree in wild type s ib l ings. Also absent were the terminal end buds seen during normal ductal morphogenesis. These findings demonstrate that the absence of functional estrogen receptor is not le tha l . Although the reproductive capabi i t ies have been al tered, prenatal development of the reproductive t racts of both sexes appears to be independent of estrogen receptor medicated responses. This animal should provide an ef fect ive in vi~o model to discern the role of ER in a var ie ty of physiological processes.

Influence of light dark cycles on estradiol-17 beta induced luteinizing hormone patterns of the prepuberal gilt.

Two groups of Yorkshire gilts (110 d of age) were maintained in two light regimens. Both light regimens consisted of 14 h of light and 10 h of darkness, but were 180 degrees out of phase. Gilts in Group 1 received light from 1200 to 0200 and gilts in Group 2 from 2400 to 1400. At approximately 140 d of age each group was divided into four subgroups of eight gilts each (1A, 1B, 1C, 1D or 2A, 2B, 2C, 2D). All gilts were blood sampled at 2-h intervals for 5 d commencing on d 142. The four subgroups received a single injection of estradiol (15 micrograms/kg body weight) on d 143 at either 2400 (A), 0600 (B), 1200 (C), or 1800 (D). For pigs in Groups 1A and 1D, the injection of estradiol coincided with the animals' "subjective day" and the injections given to Groups 1B and 1C with their "subjective night." When estradiol-17 beta (E2) was administered to the gilts during their subjective day the LH profile showed one peak, whereas when E2 was administered during dark hours the profile exhibited two peaks (P < .0001). In Group 2 for which the light cycle was reversed, the well-defined spikes of LH were found to coincide with the injections of estradiol administered during the dark hours. Smaller biphasic peaks of LH occurred when injections of estradiol coincided with the light hours.(ABSTRACT TRUNCATED AT 250 WORDS)