Temporal response of uterine prostaglandins to estradiol treatment in the ovariectomized-prenant rat

Previous studies in our laboratory have shown that 24 hours of estradiol treatment significantly enhanced uterine prostaglandin (PG)F, PGE and thromboxane B₂ (TxB₂) leels but had no effect on 6-Keto-PGF_1α (6KF) concentrations in ovariectomized-pregnant rats. One explanatior for the lack of an augmentation in 6KF was a temporal differences in response (i.e. 6KF increased and decreased within the 24 hour period). To test this possibility rats were ovariectomized on day 19 of pregnancy and sacrificed 0, 4, 8, 12, 16, 20 and 24 hours after estradiol treatment. Uterine tissue and venous plasma were analyzed for PGs by radioimmunoassay. No significant (p > .05) alterations were detected for any of the uterine PGs at 0, 4, 8 and 12 hours. However, at 16 hours PGF, TxB₂ and PGE all showed significant (p > .05) increases (2.4, 3.4 and 2.1 fold, respectively) compared to 12 hours. In contrast, no significant augmentation in 6KF levels (p > .05, 1.3 fold) was detected at 16 compared to 12 hours although it was enhanced relative to 0 and 4 hours. In addition, PGF, TxB₂ and PGE, but not 6KF, showed further increases 24 hours after estradiol administration. No alterations were found (p > .05) for any of the PGs in uterine venous plasma at the time points studied. In summary, uterine PGF, PGE and TxB₂ net production appears to be more enhanced by estradiol treatment than 6KF at the time points studied. In addition, there is a slight, but significant, difference in the temporal response characteristics of 6KF compared to the other PGs. The data suggest that the dramatic increase in uterine PGF, PGE and TxB₂ levels at parturition in the rat are probably significantly related to enhanced levels of estradiol. However, the majority of the increase in uterine 6KF levels at labor is more likely caused by factors other than augmented plasma estradiol.     

Oviduct response to prostaglandins: Influence of estradiol and progesterone

Oviductal motility was measured in the isthmus of ovariectomized New Zealand rabbits. The effects of estradiol and progesterone on spontaneous motility and on the response of the oviduct to exogenously administered prostaglandin E₁ (PGE₁) and PGF_2α were determined. Estradiol treatment significantly increased both the amplitude (P<0.05) and frequency (P<0.01) of spontaneous contractions. The amplitude of spontaneous activity was less following progesterone treatment than following estradiol treatment (P<0.05). Progesterone treatment increased the duration of oviduct response to PGE₁ (P<0.05). Estradiol treatment had no effect on the response to PGE₁. Increased oviductal activity caused by PGF_2α lasted significantly (P<0.01) longer in ovariectomized, untreated animals than in ovariectomized animals treated with estradiol or progesterone. Progesterone was more effective than estradiol in decreasing the duration of the response to PGF_2α. These effects of steroid hormones on the responsiveness of the oviduct to PGE₁ and PGF_2α could contribute to the physiological control of egg transport. The nadir of ovarian hormone influence, as in the recently ovariectomized animals and as occurs immediately after ovulation, is associated with a high responsiveness of the oviduct to PGF_2α. This could effectively increase isthmic occlusion and prevent the eggs from passing through the oviduct prematurely. The gradual increase in ovarian estradiol and progesterone secretion during the 3 days following coitus could result in decreased responsiveness to PGF_2α and increased responsiveness to PGE₁. These changes might cause relaxation of isthmic tone and allow movement of eggs through the isthmus into the uterus.     

response of relaxin-treated rat uterus to prostaglandins and oxytocin

Paired segments of rat uterus were treated with relaxin (W1164-3, 150 GPU/mg) until the amplitude of contraction was reduced to at least 50% of the pre-treatment amplitude. Test segments then received 100 ng of either PGE₁, PGE₂, PGF_2α or 250 uU of oxytocin. Control segments remained untreated. There was a significant increase in contraction amplitude in response to the spasmogens (P < 0.05) but no increase was seen in controls.     

Differentiation of estradiol receptors in rat uterine cytosol by sensitivity to tamoxifen.

Analysis of rat uterine cytosol by means of immobilized antibody discloses the presence of two distinct high affinity, low capacity estradiol binding components. One of these is readily saturable by the antiestrogen tamoxifen (TS = tamoxifen sensitive), the other is tamoxifen insensitive (TI). Only TS-estradiol binding shows positive cooperativity at low ligand concentration. TS but not TI is lost when frozen tissue is thawed at 4°C then refrozen and stored for an additional 12 hours. Experiments with ovariectomized rats show that TI is formed only in the presence of estradiol. In the estrus cycle TS increases in the order: metestrus, diestrus, proestrus and estrus. The quantity of TI is the same in metestrus and diestrus and also the same but fractionally higher in proestrus and estrus.     

Discrete early changes in cellular subpopulations of rat uterine and anterior pituitary estrogen receptors in response to acute exposure to exogenous estradiol

Distribution of estrogen receptors among ligand-occupied and unoccupied species in cytosolic and nuclear subcellular compartments has been analyzed as an acute response to administration of 5 micrograms of estradiol in adult female rats. Patterns of anterior pituitary and uterine receptor turnover were monitored at intervals over a 5-h period, using either intact or 2-weeks ovariectomized animals. In terms of total cellular receptor content, initial levels were higher in castrate animals, but rapidly fell to intact levels within an hour following estradiol injection. Cycloheximide given shortly before estradiol had no effect on total pituitary receptor patterns, but appeared to result in an elevation in total uterine receptor content at early intervals. Unoccupied cytosol receptors were rapidly depleted and, with the exception of castrate pituitary samples, showed some replenishment within 5 h, all of which was cycloheximide-sensitive. Initially, occupied cytosol receptors were low in intact rats, but were present at levels approaching those of the unoccupied cytosol receptor forms in the ovariectomized rat tissues. Occupied cytosol receptor levels fluctuated in response to estradiol. Subpopulations of nuclear receptors, especially the unoccupied species, showed significant tissue specificity. In the uterus, unoccupied nuclear forms were initially present in high amounts, and the levels did not change in response to estradiol administration. In the pituitary, the levels of these receptors rose and subsequently fell over the 5-h interval. Cycloheximide conferred a similar biphasic response to estradiol upon the otherwise insensitive unoccupied nuclear forms of the uterus. Occupied nuclear receptors turned over completely during the 5-h study interval, with the kinetics being faster in the castrate than the intact tissues. Cycloheximide affected occupied nuclear forms of the uterus only, dramatically increasing their levels in response to estrogen and causing prolonged retention in the castrate animal model. Collectively, the cycloheximide effects on this system are consistent with early estrogen induction or stimulation of a protein which inhibits accumulation of occupied or unoccupied receptor species within the nucleus. This re-examination of all forms of cellular estrogen receptors as they fluctuate acutely in response to exogenous estrogen has revealed several heretofore undetected responses which must be incorporated into the overall scheme of early estrogen action.     

Inhibition of rat uterine estradiol receptor by aurintricarboxylic acid

Estradiol-receptor complex from rat uterus has been shown to have an affinity for DNA-cellulose and ATP-Sepharose. This DNA and ATP binding of estradiol receptor was observed to be sensitive to low concentrations (0.01–0.2mM) of aurintricarboxylic acid. The inhibitor was more effective when added to preparations that contained activated estradiol-receptor complex. Steroid binding properties of the receptor remained intact under the above conditions as judged by charcoal adsorption assays and sucrose gradient analysis. In addition, a 40% inhibition in the nuclear translocation of cytosol estradiol receptor was observed when rat uteri were incubated with 10nM [³H] estradiol under an atmosphere of 95% O₂ and 5% CO₂ in the presence of aurintric-carboxylic acid. Our results suggest that aurintricarboxylic acid is an effective inhibitor of rat uterine estradiol receptor and that it may be acting by interfering with site(s) on the estradiol receptor which may be exposed upon activation and are subsequently involved in processes such as ATP binding, nuclear uptake and DNA binding.     

In vitro response of relaxin-treated rat uterus to prostaglandins and oxytocin

Paired segments of rat uterus were treated in vitro with relaxin (W1164-3, 150 GPU/mg) until the amplitude of contraction was reduced to at least 50% of the pre-treatment amplitude. Test segments then received 100 ng of either PGE1, PGE2, PGF2alpha or 250 uU of oxytocin. Control segments remained untreated. There was a significant increase in contraction amplitude in response to the spasmogens (P less than 0.05) but no increase was seen in controls.     

Effects of estradiol and progesterone on uterine prostaglandin levels in the pregnant rat

Prostaglandin D₂ was found to be a potent inhibitor of B-16 melanoma cell replication $\text{in vitro}$. The inhibition was dose-dependent between 3×10^?9M and 3×10^?6M (IC_50～ 0.3 μM after 6 days). On a molar basis, PGD₂ was a better inhibitor than PGA₂ or 16,16-dimethyl-PGE₂-methyl ester (di-M-PGE₂) and in higher concentrations (10^?6?10^?7M), comparable to retinoic acid. In higher concentrations, PGD₂ inhibited DNA, RNA and protein synthesis. The B-16 melanoma cell line which we used synthesized arachidonic acid metabolites which comigrated with PGA₂, PGD₂, PGE₂ and PGF_2α on a thin layer chromatography system.     

Concepts related to salt resistant estradiol receptors in rat uterine nuclei: nuclear matrix

When ³H-estradiol (0.1 μg) is injected into immature female rats, virtually all of the label that is recovered with uterine nuclei can be solubilized by 0.6 M KCl. Salt resistant uterine nuclear estrogen binding sites do not become labeled within one hour after the injection of ³H-estradiol, but these sites do exist and can be revealed when isolated nuclei are subjected to an $\text{in vitro}$ estradiol exchange assay. These saturable, high affinity salt resistant sites appear to be associated with the uterine nuclear matrix, a residual structure of the nucleus.     

Postnatal development of uterine response to estradiol-17β in the rat

The gradual acquisition of uterine responsiveness to a single injection of estradiol-17β was studied in rats aged 5–30 days with respect to wet weight and the content of DNA, RNA, and protein. No significant response in any of these parameters was found in 5- or 10-day-old rats. In 15- and 20-day-old rats all the parameters, except DNA content, were elevated by the hormone treatment; only in 30-day-old rats was there a significant increase in DNA content.     

Tyrosinase-like activity and estradiol binding in rat uterine nuclear extracts.

Nuclear extracts from the uteri of estradiol-implanted rats contain a tyrosinase-like enzyme that has three activities: monophenolase or cresolase, diphenolase or catecholase, and estrogen binding. When [3H]estradiol was used as a substrate, 3H2O was released from the A ring in the presence of copper and ascorbic acid. The optimal concentrations of these cofactors for the cresolase activity were established. The cresolase activity was lost on attempts at further purification. Estradiol binding was observed in conjunction with the enzymatic activity and was dependent on the presence of ascorbic acid and copper. The most potent inhibitors of 3H2O release from [3H]estradiol were those with a dihydroxyphenol moiety. The reaction was also sensitive to sulfhydryl reagents. These features of the enzyme are distinctive from other oxidases capable of attacking the aromatic ring of estrogens.     

Effect of estradiol on estrogen receptor expression in rat uterine cell types

In rodent uterus, both up- and down-regulation of estrogen receptor alpha (ERalpha) messenger ribonucleic acid (mRNA) and protein levels by estradiol has been demonstrated; however, it is not known which of the uterine compartments (endometrial epithelium, stroma, myometrium) respond to estradiol with autoregulation of ERalpha. The purpose of the present study was to investigate and compare the kinetics and cell type-specific effects of estradiol on uterine ERalpha expression in immature and adult rats. Ovariectomized female rats were injected s.c. with sesame oil or estradiol-17beta. Uteri were collected and analyzed for changes in ERalpha mRNA using RNase protection assays (RPA) and in situ hybridization using radiolabeled probes specific for ERalpha. Immunohistochemical analysis was performed with a polyclonal antibody specific to ERalpha. Expression of ERalpha in the uterine epithelial cells decreased at 3 and 6 h after estradiol administration to immature and adult rats, respectively. At 24 h, ERalpha mRNA levels in the immature and mature rat uterus were higher than pretreatment levels but returned to baseline by 72 h. Pretreatment with cycloheximide did not block the 3-h repressive effect of estradiol, suggesting that the estradiol-induced decrease in ERalpha mRNA occurs independent of new protein synthesis. A decrease in ERalpha mRNA and protein was also observed in uterine epithelia at 3 and 6 h after an estradiol injection to immature and adult rats, and intensity of both the in situ hybridization signal and the immunostaining in the epithelium increased at 24 and 72 h. However, the periluminal stromal cells in the adult uterus and the majority of stromal cells of the immature uterus appeared to have increased ERalpha expression. The results indicate that down-regulation of ERalpha in the epithelia and up-regulation of stromal ERalpha play a role in early events associated with estradiol-induced cell proliferation of the uterine epithelia.     

Binding of 17-alpha-ethinyl estradiol and mestranol to uterine and pituitary gland receptors of the rat in vitro

Comparative study, through radiochemical analysis, of the binding of estrogens to the uterine and pituitary receptors in immature rats showed that, for binding to the uterine recpetors, a free OH group in Position 3 is necessary. 17alpha-ethinyl estradiol as well as mestranol (17alpha-ethinyl estradiol-3-methyl ether), showed a stronger affinity for the pituitary than the uterine receptors. Indeed, mestranol, among the 3-methyl ether estrogens, has a special place in respect to its affinity for the pituitary. Since the binding to the receptors is an essential stage in inhibition or stimulation effects, this is a significant point. It may be that mestranol exercises its recognized central effect not only indirectly through release of ethinyl estradiol, but also directly.     

Effects of estradiol and progesterone on rat uterine ribonuclease inhibitor activity

The effects of estradiol (E2) and progesterone on rat uterine RNase inhibitor activity were investigated. RNase inhibitor activity was found to be most prominent in nuclear and cytosolic fractions of the uterus. Uterine RNase inhibitor activity in the 45,000 x g cytosolic fraction was stimulated by E2 over a 6-24 hr treatment period. Progesterone did not produce a significant change in the uterine cytosolic RNase inhibitor activity during the same period.