Surface behaviour of oestradiol-17beta.

The absorption of [3H]oestradiol-17beta from its aqueous solutions has been measured in the range 0-10mug/ml. It is found that the first adsorbed molecules are parallel to the interface and occupy 100 A2. Those adsorbed in the range 6-10 mug/ml occupy 21 A2. They are presumably associated. When the adsorption occurs in the presence of a synthetic lecithin monolayer, the molecular area is equal to 16 A2. Surface tension measurements of the solutions of oestradiol-17beta and a parallel study of their fluorescence have been performed. No association of the hormone molecules has been observed in bulk. It is concluded that surfaces and liquid monolayers may favour molecular association of the oestradiol-17beta.     

Active immunization of the cow against oestradiol-17beta

Six beef heifers were immunized over a 4-month period with an oestradiol-17beta-BSA conjugate in Freund's adjuvant. There was an interference with oestrus in the treated heifers; 2 ceased to exhibit oestrus, one exhibited one oestrus and three exhibited oestrus after Day 47 of treatment. The control heifers treated with Freund's adjuvant had normal oestrous cycles. The antiserum titre rose in all treated heifers and attained its highest level in the 2 animals in which oestrus did not recur. The temporal changes in plasma LH, progesterone and oestradiol were normal during the pretreatment period, but became abnormal during the 120 days after immunization. Although plasma oestradiol-17beta rose at the expected time of oestrus after treatment, it was apparently effectively neutralized by the antiserum induced by treatment as evidenced by the absence of an LH surge. Plasma progesterone levels fell to baseline and remained low, indicating lack of formation of corpora lutea.     

Phosphorylation on tyrosine of oestradiol-17 beta receptor in uterus and interaction of oestradiol-17 beta and glucocorticoid receptors with antiphosphotyrosine antibodies

In whole rat uterus incubated in the presence of [32P]orthophosphate the oestradiol receptor is [32P]phosphorylated on tyrosine. This finding follows our previous observation that in vitro this receptor can be phosphorylated on tyrosine by a uterus kinase that endows the receptor with oestradiol-binding activity. The calf uterus oestradiol receptor interacts with high affinity with 2G8 and 1G2 antiphosphotyrosine antibodies coupled to Sepharose (Kd values of 0.28 and 1.1 nM, respectively). The interaction with 2G8 antibody has been exploited to purify the oestradiol receptor. This interaction disappears after inactivation of the oestradiol receptor by the nuclear phosphatase that hydrolyses phosphotyrosine of the receptor. This fact substantiates the evidence that the oestradiol receptor in uterus is phosphorylated on tyrosine and that this phosphorylation is required for hormone binding to the receptor. The rat liver glucocorticoid receptor also interacts with high affinity with 2G8 antiphosphotyrosine antibody coupled to Sepharose (Kd value of 0.21 nM). This receptor has been purified by using in sequence heparin-Sepharose and antiphosphotyrosine antibody-Sepharose.     

The histochemistry of oestradiol-17 beta and isocitric dehydrogenases in endometrial carcinoma

Specimens of endometrial carcinoma were obtained from 8 women, four of whom had previously been treated with oral medroxyprogesterone acetate 200 mg daily for 7 days. The activities of oestradiol-17 beta and isocitric dehydrogenases and nuclear oestradiol receptor concentrations were measured in the homogenised tissue and both enzymes were located histochemically. Histochemical evidence of oestradiol dehydrogenase activity was found in all but one of the specimens with biochemical activity. This anomalous specimen was obtained from a woman who had not been treated with MPA and whose endometrium exhibited only low levels of enzyme activity. The histochemical staining caused by isocitric dehydrogenase was intense but bore no relation to the biochemical measurement of enzyme activity in the homogenate. The modified technique for the histochemical demonstration of oestradiol dehydrogenase activity although not quantitative gave results similar to the biochemical methodology. It may therefore be useful as a simple test of the prediction of the sensitivity of endometrial carcinoma to progestogens.     

Protein synthesis in the uterus of the immature rat: the effects of short exposures to oestradiol-17 beta

Three methods were used to study the effect of oestrogen on the incorporation of radioactive precursor into uterine protein. Intact tissue was incubated in vitro. Isolated uterine epithelial, stromal and myometrial cells were labelled in vitro. Isolated polysomes were translated in cell free protein synthesising systems. In all of these systems, minor qualitative changes in protein synthesis were detected where the uteri were derived from oestrogen-treated rats. These changes were most dramatic in isolated stromal cells and were accompanied by a marked overall increase in protein synthesis. The translation of mRNA on isolated uterine polysomes revealed a sequence of minor, but reproducible, oestradiol-induced changes. It was difficult, however, to relate these changes to those detected in incubated tissue or cells, possibly because the cell free translation products were not subject to normal post-translational modification and processing.     

Metabolism of oestradiol-17beta by intestinal bacteria in rats.

In vitro experiments were carried out in which [4-14C]oestradiol-17beta was incubated with a culture from caecal content from adult male rats at 37 degrees C in an atmosphere of nitrogen. Oestrone was identified as the only certain metabolite. Other metabolites, if present, were quantitatively unimportant. The conversion of oestradiol-17beta to oestrone was estimated to be 22-42%.     

Differential subnuclear distribution of polyadenylate-rich ribonuclei acid during induction of egg-yolk protein synthesis in male Xenopus liver by oestradiol-17 beta.

A 4-8-fold increase in the rate of hepatic nuclear RNA synthesis occurred within 11 h after a single injection of oestradiol-17 beta to male Xenopus to induce egg-yolk protein synthesis. 2. By using a gentle procedure for fractionating nuclei into their major structurally different components [J. R. Tata& B. Baker (1974) Exp. Cell Res. 83. 111-124], it was found that the hormone-induced increase in the total amount of newly made RNA was associated with a 2-10-fold increase in the poly(A) content of nuclear RNA. 3. When the poly (A) content of nuclear RNA was determined by hybridization to poly[3H](U) or specific binding to oligo(dT)-cellulose, most of the increase (10-fold) in poly (A) content of newly synthesized RNA was associated with the euchromatin fractions, whereas the increase was less marked in the other subnuclear fractions. 4. Resolution of nuclear RNA into poly (A)-poor and poly(A)-rich RNA species by chromatography on oligo(dT)-cellulose, followed by polyacrylamide-gel electrophoresis with sodium dodecyl sulphate or in the pressence of 99% formamide, revealed that the hormone caused a preferential enhancement of high-molecular-weight (25S-60S) poly (A)-rich HnRNA (heterogeneous nuclear RNA,) much of which was associated with euchromatin and not with the nuclear sap. 5. Induction of vitellogenin in male frogs was in particular characterized by the appearance of a high-molecular-weight polyadenylated component exhibiting a peak at 35-36S, i.e. a molecular weight of approx. 2.05x10(6)+/-0.15x10(6). Although there is no evidence as yet that such a polyadenylated high-molecular-weight nuclear RNA species contains sequences corresponding to vitellogenin mRNA, it is possible that a high proportion of the most stable form of the putative nuclear precursor to vitellogenin mRNA induced by oestrogen in male Xenopus liver may be only marginally bigger than the cytoplasmic mRNA, and may at any one time be predominantly associated with the euchromatin fraction.     

Pituitary response of prepuberal lambs to oestradiol-17 beta.

In two experiments 48 prepuberal Merino ewe lambs were injected with oestradiol-17 beta (E2) or saline to study the effect of E2 on their plasma LH levels and on oestrus and ovulation. In the three groups which received 30 (experiment I), 50 and 30 (experiment II) microgram E2 respectively, 27 out of 28 lambs showed an LH response, the corresponding mean LH peaks being 64.3 +/0 22.5, 153.6 +/-33.4 and 91.7 +/- 16.9 ng/ml at mean intervals of 11.1, 11.2 and 10.5 h, respectively, after injection. None of the 20 lambs in the control groups had an LH level higher than 18 ng/ml 12 h after injection. In the three E2 groups, 41.7, 62.5 and 37.5% of animals showed oestrus within 26 h of injection while in the control groups only one animal showed oestrus. Of 13 animals showing oestrus in the E2 groups, 11 failed to ovulate. The mean pre-injection plasma FSH level in experiment I was 102.7 ng/ml, and in four 5--7-month-old lambs over several weeks uas 155.3 ng/ml. Despite these high pre-injection levels of FSH, it appears that the follicles were unable to respond to the LH peak which followed the E2 injection.     

Characterization of uterine heterogeneous nuclear ribonucleic acid and the effect of oestradiol-17 beta on its synthesis.

An early response to the administration of oestradiol-17 beta to immature rats is a dramatic stimulation in the synthesis of uterine hnRNA (heterogenous nuclear RNA). High-molecular-weight fractions of the hnRNA were purified and subfractionated on poly(U)-Sepharose into fractions that differed in their poly(A) content and their size profile on polyacrylamide gels. Oestrogen treatment of the rats stimulated the synthesis of all three fractions of high-molecular-weight hnRNA, but the kinetics of synthesis, degree of stimulation and size distribution of the newly synthesize RNA differed in each fraction.     

Persistent infertility in ewes after prolonged exposure to oestradiol-17 beta

Merino ewes were treated with implants which released 300 micrograms oestradiol-17 beta per day or 5 mg progesterone per day, or both, for 9 months (Months 1-9), and after an 11-month intermission were treated again for 6 months (Months 20-26). Ewes were run with rams at Months 16, 28 and 40. Fertility was not affected by the first exposure period, but the second exposure to oestradiol reduced the fertility of ewes at both subsequent mating periods. Affected ewes returned to service more frequently (P less than 0.01) and were less likely to conceive (P less than 0.05). After mating, a normal population of spermatozoa was established in the caudal cervix, but transport through the cervix was impaired in affected ewes and there were fewer spermatozoa (P less than 0.01) in the cranial cervix. In affected ewes, the spinnbarkeit of cervical mucus was reduced (P less than 0.05), and the histological appearance of the cervix changed, looking like that of the uterus. Treatment with progesterone did not affect fertility, cervical mucus or sperm transport, but diminished the histological abnormalities produced by oestradiol (P less than 0.05). These results show that oestradiol-17 beta given after puberty can cause the same kind of permanent sexual transdifferentiation that is produced by the oestrogenic isoflavones in ewes with clover disease. The results suggest that this change may require more than a single exposure to oestrogen.