Contrasting effects of estradiol-17 beta and 17 alpha-ethinyl estradiol-17 beta on cultured whole embryos

Estradiol-17 beta (E2) and 17 alpha-ethinyl estradiol-17 beta (EE) were compared in terms of their relative capacities to alter growth and developmental patterns of cultured whole embryos during the early stages of organogenesis. Embryos exhibited a notable differential susceptibility to the embryotoxic effects of parents E2 vs EE when these estrogens were added directly to the media at the onset of the culture period. At initial concentrations of 0.1 mM, E2 failed to produce statistically significant effects whereas EE elicited marked embryotoxicity. Inclusion of a P-450-dependent biotransformation system in the culture media resulted in a significant attenuation of the embryotoxic effects of parent E2 vs EE when these estrogens were added directly to the media at the onset of the culture period. At initial concentrations of 0.1 mM, E2 failed to produce statistically embryotoxicity by hepatic S9. The divergent results produced by the two steroids could not be attributed to differences in rates of catecholestrogen generation in the culture medium or by the conceptuses. The results demonstrate definitive dissimilarities between the effects of two steroidal estrogens on developmental parameters and document marked differences in the effects of biotransformation on their embryotoxic potential. The data strongly suggest that the embryotoxicity of these steroids is not mediated via interactions with estrogen receptors. Additionally, the data show that the differential capacity of these two steroids to produce embryotoxic effects is diametrically opposite to earlier reported patterns of their carcinogenic potential in the Syrian hamster kidney.     

Regulation of protein synthesis by estradiol-17beta in cultured hepatocytes

Estradiol-17beta added to cultured chick embryo hepatocytes induced the appearance in the medium of a phosphoprotein, identified as phosvitin on the basis of: (i) its behaviour on ionic exchange columns; (ii) its SDS-acrylamide gel electrophoretic mobility; (iii) its amino acid composition. The hormone treatment was also followed by a decreased synthesis of other proteins secreted by the hepatocytes.     

Action of estradiol-17 beta, estrone sulfate and progesterone on the growth and differentiation of endometrial epithelial cells of the guinea pig in primary culture

Endometrial guinea-pig glandular epithelial cells grown in primary culture incorporated [3H] thymidine. After three washings with a steroid-free medium, they were made quiescent and arrested in the G0/G1 phase. However, they remained hormone-responsive and resumed the cellular cycle after stimulation by 10(-6) M oestrone sulphate but not by oestradiol-17 beta.     

Plasma estradiol-17beta level in the domestic fowl

Using healthy white Leghorn chickens the estradiol-17β level in plasma were determined by radioimmunoassay and the dependency on age and sex examined. In one and two year old laying hens average values between 48.0 and 54.4 pg/ml were found. The estradiol level in cocks of the same age were between 6.0 and 7.3 pg/ml. Significant difference with respect to the estradiol level were already noticeable in sexually immature 4 month old chickens ♀: 8.1 pg/ml, ♂: 1.0 pg/ml). The significance of the estradiol-17β level with respect to plasma lipids and electrophoretic mobility of serum lipoprotein is shown.     

Formation of glucuronides of estradiol-17 beta by human mammary cancer cells

The formation of glucuronides of estradiol-17 beta by human mammary cancer cell lines is reported for the first time. When incubated with [3H]estradiol-17 beta (1 nM) for 16 h, ZR-75-1 and T47-D cells formed estradiol-3-glucuronide and estradiol-17 beta-glucuronide in approximately equal proportions, whereas MCF-7 cells formed E2-3-glucuronide only. Yields of monoglucuronides from MCF-7 and ZR-75-1 cells were 0.35 pmol/mg DNA, which represented 20-26% of the yield of estradiol-monosulphates. A HPLC system capable of separating most estradiol monosulphates, monoglucuronides and mixed conjugates, is described.     

Genotoxic effects of estradiol-17beta on human lymphocyte chromosomes

The cytogenetic effect of a hormonal steroid, estradiol-17beta, was assessed in peripheral blood human lymphocyte culture. Sister chromatid exchanges (SCE) and chromosome aberrations (CA) were scored as genetic end points. Significant induction of CA was observed at 25 microg/ml and 50 microg/ml concentrations of estradiol-17beta in the absence of microsomal activation. The drug was effective in all treatments in the presence of rat liver S(9) microsomal fraction (S(9) mix) and exhibited increased frequency of chromosomal aberrations. The drug was effective in increasing the SCE frequency which was found to be maximum at the dose of 50 microg/ml concentration (i.e., 4.34+/-1.22) both with and without metabolic activation. It was found that estradiol-17beta itself and possibly its metabolites are potent mutagens beyond a particular dose in human lymphocytes.     

Cytokine-induced production of IFN-beta 2/IL-6 by freshly explanted human endometrial stromal cells. Modulation by estradiol-17 beta

The cytokine IFN-beta 2/IL-6 has emerged as an important means of communication between cells--both within the immune system as well as outside it. In exploring the link between the endocrine and the immune systems, we have studied the secretion of IFN-beta 2/IL-6 by freshly explanted human endometrial stromal cells and its modulation by estrogens. Endometrial stromal cells produced IFN-beta 2/IL-6 in response to other inflammation-associated cytokines such as IL-1 alpha or beta, TNF, and IFN-gamma. This secretion was strongly inhibited by estradiol-17 beta at concentrations as low as 10(-9) M. Multiple species of stromal cell IFN-beta 2/IL-6 in the size range 23 to 30 kDa were detected using immunoprecipitation or immunoblotting procedures. The endometrial stromal cell IFN-beta 2/IL-6 species were phosphorylated and differentially glycosylated in a manner comparable to IFN-beta 2/IL-6 secreted by induced human peripheral blood monocytes or foreskin fibroblasts. However, in contrast to peripheral blood monocytes and fibroblasts, bacterial LPS did not induce IFN-beta 2/IL-6 production in endometrial stromal cells. Additionally, the IFN-beta 2/IL-6 identified in medium from IL-1 alpha-induced stromal cells is biologically active on hepatocytes. These observations, taken together with the observation that IFN-beta 2/IL-6 strongly inhibits the proliferation of human epithelial cells, suggest the possibility that stromal cell secreted IFN-beta 2/IL-6 may affect the physiology of the overlying epithelium in an hormonally modulated manner. Estrogen-regulated production of endometrial IFN-beta 2/IL-6 may participate in gender-specific systemic immunomodulation.     

A study of the source of estradiol-17 beta in the bull

A study was designed to determine if the bull testes secretes estradiol-17 beta, as has been reported for several other species. Two experiments were conducted. In the first experiment, five Angus-sired crossbred bulls were fitted with catheters in the spermatic and jugular veins and sampled every 15 min for six hours. One was bled from three cannula, the third being in the spermatic artery. In the second experiment, these same bulls were castrated and jugular vein blood was collected at timed intervals for two hours. Plasma samples were assayed for estradiol-17 beta (E2) and testosterone (T) using highly specific radioimmunoassays. This experiments gave evidence that: a. Concentrations of E2 were significantly lower (P less than .05) in the jugular vein than the spermatic vein in each of the five bulls, although the mean concentration for all bulls was not great for either vein nor was the difference great between the two veins. In confirmation of past studies, T concentration in the jugular vein was much lower than in the spermatic vein in each bull as was overall mean. b. Removal of the testes caused E2 to decrease during the 25 min post-castration but the difference was not significant (P greater than .05), whereas T decreased 4-fold (P less than .01). During the next 95 min., the concentrations of both hormones increased 3- and 6-fold, respectively, as did cortisol concentration. It is concluded that the bull testes secretes E2, but the secretion is minor to that of T and that another source of both hormones can be the adrenal gland, such as during stress.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endometrial expression of cellular protooncogene c-ski and its regulation by estradiol-17beta.

The expression of the cellular protooncogene c-ski was examined in the rat uterus. In situ hybridization revealed that c-ski mRNA was expressed in the uterus of the adult rat on the day of estrous and localized mainly in the luminal and glandular epithelia. To test the possibility that the expression of c-ski mRNA is induced by estrogen, rats were ovariectomized and estradiol-17beta (E2) was injected. The expression of c-ski mRNA was upregulated 3 h after E2 treatment, reaching the highest level at 6 h and this persisted until 24 h; the E2-induced expression of c-ski mRNA was restricted to the luminal and glandular epithelia. These results suggest that the c-ski gene plays a role in uterine epithelial cell proliferation and mediates the proliferative action of E2.     

Direct regulation of beta3-integrin subunit gene expression by HOXA10 in endometrial cells

Estrogen and progesterone regulate HOXA10 expression in the endometrium, where HOXA10 is necessary for implantation. The integrins are also involved in early embryo-endometrial interactions. Here we show that HOXA10 directly regulates beta3-integrin subunit expression in the endometrium, likely mediating the effect of sex steroids on beta3-integrin expression. beta3-Integrin expression was decreased in endometrium shown to have low HOXA10 expression. beta3-Integrin mRNA levels were increased in endometrial adenocarcinoma cells (Ishikawa) transfected with pcDNA3.1/HOXA10, and decreased in cells treated with HOXA10 antisense. Seven consensus HOXA10 binding sites were identified 5' of the beta3-integrin gene. Direct binding of HOXA10 protein to four sites was demonstrated by EMSA. Reporter gene expression increased in BT-20 cells cotransfected with pcDNA3.1/ HOXA10 and pGL3-promoter vector containing region F (encompassing all seven HOXA10 consensus sites). A 41-bp segment (Region A) showed highest affinity binding to HOXA10 protein. Increased reporter expression, equal in magnitude to that obtained with Region F, was obtained with Region A. HOXA10 protein binding within Region A was localized by deoxyribonuclease I footprinting. beta3-Integrin expression was directly up-regulated by HOXA10 through a 41-bp 5'-regulatory element. Sex steroids regulate the expression of endometrial beta3-integrin through a pathway involving HOXA10.     

Positive and negative effects of estradiol-17 beta in the rat uterus

Estrogens could act as effectors or inhibitors of protein synthesis in the rat uterus, depending on the doses given to animals. A single injection of estradiol-17 beta to immature female rats led to the increase in protein synthesis and in enzyme activities involved in DNA synthesis. Four injections, given once daily, resulted in the inhibition of enzyme activity and synthesis of all proteins but one. The 105 kD protein which showed a gradual increase with the duration of estrogen treatment could be responsible for the negative action of estrogens on uterine growth.     

Effect of estradiol-17 beta and prostaglandins on rat myometrial gap junctions

The effects of estradiol-17 beta and indomethacin on myometrial gap junction development, plasma estradiol levels and uterine PGF2 alpha content were evaluated in immature and/or ovariectomized, mature rats. High doses of estradiol stimulated the development of gap junctions in the myometrium of animals from both groups. Concomitant injections of estradiol and indomethacin to ovariectomized rats potentiated the estradiol stimulation of gap junctions. Plasma estradiol levels were lower in ovariectomized rats treated with both estradiol and indomethacin than in animals treated with estradiol alone. Indomethacin also enhanced the uptake and retention of 3H-estradiol into uterine tissues. Uterine PGF2 alpha content of ovarectomized rats was stimulated with the initial injection of estradiol but thereafter, the PGF2 alpha content declined with repeated injections to values lower than that observed in controls. Prostaglandin F2 alpha content in tissues from rats treated with estradiol plus indomethacin were also higher than that observed in rats treated with indomethacin alone, however, the values obtained in both groups were significantly lower compared to those from control animals. These results are consistent with the hypothesis that steroid hormones and prostaglandins regulate myometrial gap junction formation. Regulation of myometrial gap junctions by prostaglandins is discussed with respect to a down regulation of the steroid-receptor mechanism and effects on cyclo-oxygenase or lipoxygenase products.     

A non-chromatographic radioimmunoassay of estrone and estradiol-17beta serum

A sensitive and efficient non-chromatographic procedure employing the Girard reagent and solvent-partitioning has been developed for the accurate radioimmunoassay (RIA) of estrone (E1) and estradiol-17β(E2) in a single 1.0 ml specimen of male or female serum. Using standard curves which permitted the discrimination of zero from 0.75–1.5 pg (p=0.05), the following mean procedural blanks (pg ± S.D.) were determined (1.0 ml water, n= 24): estrone, 2. 1 ± 1.1 (range 0–4.1); estradiol 1.0± 1.1 (range 0–3.6).A comparison of RIA of estrogens (1) in serum after separation by the Girard procedure and by TLC yielded correlation coefficients of 0.99 and 0.98 for E1 and E2 respectively. The following results (pg/ml ± S.D.) were obtained on RIA of E1 and E2 in 12 different 1.0 ml specimens of male and female serum using the Girard procedure: male. E1 (32.0 ± 9.2), E2 (24.1 ± 10.9); female, E1 (108.5 ± 60.8), E2 (126.4 ± 63.2).The intra-assay variability (c.v.) was found to be 12.6% for E1 and 9.4% for E2. The interassay variability was 14.2% for both estrogens.Twenty-four assays of E1 and E2 can be completed by one person in 2 working days.     

The preparation of estradiol-17 beta sulfates with triethylamine-sulfur trioxide

Reaction of estradiol-17 beta with triethylamine-sulfur trioxide in pyridine gives exclusively monosulfation at the C17-hydroxyl group with the preparation of 17 beta-sulfooxyestra-1,3,5(10)-trien-3-ol triethylammonium salt (V). The structural assignment suggested by spectroscopic measurements was confirmed by synthetic studies. (Formula: see text) A synthesis of 3-sulfooxyestra-1,3,5(10)-trien-17 beta-ol triethylammonium salt (II) has been accomplished based on the preparation of 17 beta-formyloxyestra-1,3,5(10)-trien-3-ol (XIII). Fusion of the 3-sulfate triethylammonium salt II gives rise to the 17-sulfate triethylamine salt V. The preparation of estradiol-17 beta disulfate has also been achieved.