Cordycepin and early effects of estradiol on the immature rat uterus.

Estradiol induces the synthesis of a specific protein fraction (IP) in the uterus of the immature rat. The injection of cordycepin (3' deoxyadenosine), an inhibitor of poly A synthesis, inhibits the synthesis of IP. This fact suggests that one of the earliest effects of estrogen is the production of Hn-RNA poly-A relative to IP. Moreover, using electron microscopy, the stimulation by estradiol of the nucleolus of the immature rat uterine epithelium has been shown. Cordycepin does not affect this stimulation to any appreciable extent. Biochemical studies (incorporation of radioactive stracers into NRA, affinity chromatography on poly U-Sepharose) carried out in parallel with and under conditions comparable to those used in electron microscopy show that cordycepin does not greatly affect the increase in ribosomal RNA observed under the effect of estradiol. The blocking of IP by cordycepin and the lack of inhibition at the nucleolus level under the same conditions, show that the two early effects of the action of estrogen on the immature rat uterus are not directly correlated.     

The effect of estradiol on collagen structure and organization in the immature rat uterus

An examination of collagen ultrastructure in the lamina propria of the immature ovariectomized rat uterus revealed that a single injection of estradiol (40 micrograms/kg) produced a biphasic effect on collagen structure and organization. In saline-treated animals (controls) dense populations of collagen bundles were seen throughout the extracellular matrix (EX). Cross-sections of these bundles suggested that the bundles run parallel to the long axis of the uterus and thin filaments seemed to form cross-links between collagen fibers. In contrast, large clear spaces, collagen fragments, and loosely packed bundles of collagen were observed in the EX of animals injected with estradiol 24 hr earlier. In a time course study (0, 1, 2, 4, 24, and 48 hr), estradiol treatment altered collagen structure and organization 1 hr following administration. Collagen bundles did not appear to be as densely packed as in control tissues and large clear spaces were evident in the EX. Two hours following estrogen administration, collagen fibers appeared to be fragmented and seemed to be separating from the plasma membrane of stromal cells. Four hours following estrogen administration, large clear spaces occupied most of the EX in the lamina propria. Collagen fragments were diffusely distributed throughout the EX and small cross-sectional patches of collagen bundles were present. In 48-hr-treated animals, collagen bundles reappeared and were often closely associated with the plasma membrane of stromal cells. The collagen was not as abundant as in control animals. An overview of the cellular organization of the lamina propria revealed that stromal cells in control tissues were more densely packed than in estradiol-treated tissues (4, 24, and 48 hr) and the stromal cell size appeared to increase in hormone-treated tissues. These responses provide a good model system to study the role of estradiol in the control of collagen structure and organization in the uterus.     

Oestriol-stimulated synthesis of ribonucleic acid in the uterus of the immature rat.

Oestradiol-17beta and oestriol both sequentially stimulate the synthesis of heterogeneous nuclear RNA and rRNA in vivo. However, the stimulation by oestradiol-17beta, particularly of rRNA synthesis, is very much greater than that elicited by oestriol.     

Dissociation by colchicine of the wet weight and the cGMP responses to estradiol in immature rat uterus

We previously reported on a positive correlation between two effects of estrogen on rat uterus, namely the early increases in cGMP and in water content of the organ suggesting that they were under the control of the same hormone sensitive regulatory process or linked by a cause to effect relationship. Up to now we were unable to find experimental conditions that would dissociate the two responses. In this work, immature Wistar rats were treated with colchicine (50 micrograms/animal) given at the same time as estradiol-17 beta (1 microgram/animal) or with estradiol alone. The experiments showed: (1) that the estradiol induced increase in uterine wet weight that occurs during the first 8 h after hormone injection was completely suppressed in the presence of colchicine indicating that it might depend on an intact microtubular system and (2) that, by contrast, the estrogen-induced increase in uterine cGMP remained unaffected by the colchicine presence. These data allow to conclude that the cGMP response to estradiol can be dissociated from the wet weight response and, therefore, that it is not controlled by the latter. From this and from data in the literature the hypothesis is proposed that the increase in uterine cGMP content might trigger the wet weight response, this possibly through a positive action on some microtubular function.     

Estrogen induced expression of the C-jun proto-oncogene in the immature and mature rat uterus

The expression of the proto-oncogene c-jun in response to estradiol treatment in immature and mature rat uterine tissue was measured using a cDNA encoding the mouse c-jun proto-oncogene. This probe hybridized to a major RNA band of 2.7 kb and a minor 3.2 kb band. In Northern blots of total RNA from both immature and mature rat uteri, estradiol treatment resulted in at least a 3 fold increase in expression of the 2.7 kb band over control levels by 3 hr post injection. By 12 hr post injection, expression of c-jun mRNA had returned to control levels. A strong induction (greater than 5 fold) of c-jun mRNA expression was also observed in stroma-myometrial tissue isolated from mature rats approximately 3 hours after treatment with estradiol. The similar kinetics of induction of c-fos and c-jun emphasizes the functional significance of the fos/jun heterodimer in control of uterine cell proliferation.     

Evaluation of the gene expression changes induced by 17-alpha-ethynyl estradiol in the immature uterus/ovaries of the rat using high density oligonucleotide arrays

BACKGROUND: In a previous study, we determined the effects of 17-alpha-ethynyl estradiol (EE) on gene expression using microarrays that represented approximately 9,000 genes, which was the state of-the-art. Higher content arrays with almost double the number of genes have since become available. In order to better determine whether common sets of gene expression changes can be predictive of estrogenic activity, we have replicated the previous experiment using the more comprehensive microarray. METHODS: Immature 20-day-old Sprague-Dawley rats were exposed to 0.1, 1, and 10 microg EE/kg/day ( subcutaneously [s.c.]), for four days, dosing from postnatal day (PND) 20-23). Changes in a more comprehensive expression level of 15,923 rat annotated genes and expressed sequence tags were evaluated on PND 24. RESULTS: By comparing the response of the treatment groups versus controls using various statistical parameters, we determined that the expression of 1,394 genes showed a significant change with respect to control (p< or =0.0001), to at least one of the EE dosages. The tissues from animals exposed to 0.1 microg EE/kg/day showed changes in the expression of only 33 genes, whereas when they were exposed to 1 or 10 microg EE/kg/day, the expression of 409 and 548 genes was modified, respectively. A dose-dependent analysis indicated that 592 genes showed a robust and significant response to EE exposure (increased or decreased). Our analysis confirmed the regulation of previously identified estrogen-sensitive genes, and clearly identified novel mediators of estrogen actions, both in the uterus as well as in the ovary. CONCLUSIONS: This compendium of genes represents the largest compilation of estrogen-responsive genes that has ever been identified for the immature uterus and ovary of any species, and can be used to generate testable hypothesis to improve the understanding of the molecular pathways associated with physiological and pathophysiological responses to exposure to chemicals with estrogenic properties.     

Oestrogen-induced expression of oncogenes in the immature rat uterus

4 h after a single precocious administration of oestrogen there was a considerable but short-lived surge in the uterine levels of myc-encoded polyadenylated mRNA. This was followed by a further peak 28 h after hormone administration. The expression of rasHa showed a totally different time course with a build up of hybridizable message that peaked 8 h after oestrogen administration.     

Progesterone binding in the immature mouse and rat uterus

The use of a highly active progestin, 17, 21-dimethyl-19-nor-4, 9-pregnadiene-3, 20-dione (R 5020), as a tag has established that progesterone binds to a specific “7–8S” uterus cytosol component in both the immature mouse and rat.     

Synthesis of alpha fetoprotein by immature rat uterus

Reported free and bound molecular forms of alpha fetoprotein detected in immature uterine cytosol could be due to either selective uptake from the serum and/or intracellular synthesis by this tissue. In this study immature rat uterus synthesized initially immunounreactive bound alpha fetoprotein, which becomes immunoreactive after treatment with 0.4M KCl, but failed to synthesize free alpha fetoprotein. This indicates that bound alpha fetoprotein is not a conversion product of the free form, and suggests a relationship between alpha fetoprotein synthesis and uterine growth and development.     

Early transcriptional events induced by estradiol in rat uterus. Partial purification of specific protein fraction

The specific protein fraction induced by estradiol in the rat uterus (IP) was purified by selective acid precipitation, DEAE cellulose chromatography, Sephadex G200 filtration and preparative cellogel electrophoresis.The determination of specific phosphoprotein phosphatase activities and of the level of IP purification after the different preparation steps, confirmed Kaye's results and allowed to state that IP was not a phosphoprotein phosphatase.The purified IP preparation did not display any detectable protein kinase activity, nor estradiol complexing ability.The injection of cordycepin (3' deoxyadenosine), an inhibitor of poly A synthesis, inhibits the synthesis of IP. This fact suggests that one of the earliest effects of estrogen is the production of Hn-RNA poly-A relative to IP. Cordycepin does not greatly affect the increase in ribosomal RNA observed under the effect of estradiol.The blocking of IP by cordycepin and the lack of inhibition at the nucleolus level under the same conditions show that the two early effects of the action of estrogen on the immature rat uterus are not directly correlated.     

Concentration and turnover of estradiol in the rat uterus in vivo

The concentrations and turnover of estradiol isolated from cytosolic and nuclear fractions of uteri from ovariectomized rats given estradiol, either in single injections or in continuous infusion, were analyzed by gas chromatography-mass spectrometry. The analytical method was validated for different organs and lower limits of analysis were established. After infusion of 20 ng x h-1 for 18-22 h, mean estradiol levels were 2.0-2.4 fmol x mg-1 uterine wet weight in the nuclear fraction, and 1.2-1.5 fmol x mg-1 in the cytosolic fraction. The concentrations were about five times higher after a single injection of one microgram estradiol but the distribution between nuclear and cytosolic fractions was almost the same. The concentrations of estradiol in nuclei from liver and spleen were 50-200 times lower than those in uterus. Taken together with previous knowledge, the results indicate that the distributions of estradiol and its receptor are not the same and that hormone response cannot be predicted from the concentration of receptors alone. The exchange of estradiol molecules in the uterus was followed after a change of the infusion from unlabelled to [11,12,12-2H3]-labelled estradiol, or vice versa. The uterine uptake of estradiol was calculated to be about 0.7 fmol x h-1 x mg-1 uterine wet weight. The half-life time was calculated to be at least 4 h for estradiol molecules isolated from the nuclear fraction and 3 h (significantly shorter) for those isolated from the cytosolic fraction. The results indicate an uptake of 40-90% of all estradiol passing through the uterus in proestrus with only about 10% of available receptors becoming occupied. When the infusion was changed from estradiol to ethynylestradiol, estradiol disappeared from the uterus at the same rate as in the experiments above. Ethynylestradiol was taken up at a rate of about 0.3-0.4 fmol x h-1 x mg-1 tissue. The percentage of total steroid found in the nuclear fraction was higher for ethynylestradiol, about 70%, than for estradiol, about 60%, indicative of a more stable association of receptor to nuclear binding sites when ethynylestradiol is the ligand.     

Inhibition of oestradiol-induced DNA synthesis by opioid peptides in the rat uterus.

Opioid drugs and peptides were investigated for their effect on uterine DNA synthesis induced by a single injection of 17 beta-oestradiol given to ovariectomized rats 24 h prior to decapitation. [D-Met2,Pro5]-enkephalinamide administered 12, 2 or 1 h before killing resulted in a significant (approximately 50%) inhibition of in vitro [3H]-thymidine incorporation into DNA, while injections given 24 or 6 h before decapitation were ineffective. Non-linear regression of the dose-effect curves resulted in an ED50 of approximately 0.26 and approximately 0.45 microgram/100 g b.wt. for the opioid treatments given 12 or 2 h before killing, respectively. These effects could be completely reversed by the opioid antagonist naloxone injected 30 min prior to the agonist treatment, while naloxone itself had no effect. Morphine and [D-Ala2,D-Leu5]-enkephalin administered 12 h, as well as dynorphin A fragment 1-13 given 2 h before decapitation also inhibited oestradiol-induced uterine DNA synthesis. In ovariectomized animals without 17 beta-oestradiol priming no significant effect of [D-Met2,Pro5]-enkephalinamide or naloxone on [3H]TdR incorporation was found.