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.     

Modulation of uterine morphology and growth by estradiol-17beta and an estrogen antagonist

The estrogen antagonist C1628 maintains sustained hypertrophy of the uterine epithelium and the synthesis of many proteins including peroxidase. C1628 is a progestogen, inducing secretion of the protein by surface epithelial and glandular cells. C1628 is a connective tissue mitogen, inducing DNA synthesis in fibroblasts and the endothelium. C1628 and estrogen share these properties mentioned above. Estrogen, however, induced moderate growth of the mucosa within a 24-h period and massive hyperplasia of the mucosa within a 24-h period thereafter. C1628 alone, or in combination with estradiol, does not have mitogenic effect on the mucosa, and in fact blocks the mitotic response normally induced by estrogen alone.     

Rat brain glycolysis regulation by estradiol-17 beta.

The effect of estradiol-17 beta on the activities of glycolytic enzymes from female rat brain was studied. The following enzymes were examined: hexokinase (HK, EC 2.7.1.1), phosphofructokinase (PFK, EC 2.7.1.11), aldolase (EC 4.1.2.13), glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), phosphoglycerate kinase (EC 2.7.2.3), phosphoglycerate mutase (EC 2.7.5.3), enolase (EC 4.2.1.11) and pyruvate kinase (PK, EC 2.7.1.40). The activities of HK (soluble and membrane-bound), PFK and PK were increased after 4 h of hormone treatment, while the others remained constant. The changes in activity were not seen in the presence of actinomycin D. The significant rise of the activities of the key glycolytic enzymes was also observed in the cell culture of mouse neuroblastoma C1300 treated with hormone. Only three of the studied isozymes, namely, HKII, B4 and K4 were found to be estradiol-sensitive for HK, PFK and PK, respectively. The results obtained suggest that rat brain glycolysis regulation by estradiol is carried out in neurons due to definite isozymes induction.     

Uterine prostaglandin and blood flow responses to estradiol-17 beta in cyclic cattle

Normal cyclic dairy cattle (n = 7) underwent a midventral laparotomy on day 17 of the estrous cycle and were fitted, ipsilateral to the CL, with: an electromagnetic flow transducer around the uterine artery (UA; n = 5); catheters within the ovarian vein (OV; n = 7) via a uterine branch of the ovarian vein, uterine branch of the ovarian artery (UBOA; n = 5) and facial artery (FA; n = 7). On day 18, blood samples were collected at 30 min intervals for 1 h prior to injection of estradiol-17 beta (E2; 3 mg) and 12 h post-E2. Uterine blood flow (UBF) was monitored continuously and plasma samples analyzed for PGF2 alpha and PGFM. Exact locations of catheters in reproductive tracts were verified post-slaughter. Data were analyzed by method of least squares analysis of variance. Uterine blood flow (ml/min) increased above pre-E2 flow rates within 30 min post-E2 injection, peaked between 2.5 to 3.5 h and declined between 4 to 8.5 h. A small secondary rise in UBF occurred between 9 and 12 h. Regression analysis for concentrations (pg/ml) of PGF2 alpha and PGFM in the OV (i.e., [OV]-[FA]) demonstrate a similar response as PGFM concentration in the FA in that all increased at approximately 3 h, peaked between 5 and 7 h and returned to near baseline levels by 9 to 10 h post-E2. Facial artery PGFM concentrations were positively correlated with uterine production of PGF2 alpha (r = .66) and PGFM (r = .30), whereas FA PGF2 alpha concentrations were not. In three of five cows, a difference in PGF2 alpha was detected between UBOA and FA (UBOA greater than FA); supportive of a local countercurrent exchange between the uterine venous drainage and the ovarian artery.     

Purification and interaction with estradiol-17beta.

The combination of polyacrylamide gel electrophoresis and Concanavalin-A-Sepharose affinity chromatography has permitted the isolation on a preparative scale, of four molecular forms of rat alpha1-fetoprotein: a "slow" and a "fast" fraction, each separable into Concanavalin-A-adorbed ("high carbohydrate", i.e. rich in accessible alphaD-Mannosyl and alphaD-Glucosyl residues) and a Concanavalin-A-non adsorbed ("low carbohydrate") fractions. These four iso-alpha-fetoproteins (iso-AFP) bind estradiol-17beta. However, they disclose differences in both their association constants and number of binding sites for this hormone. Very high affinity sites (10(9)) are mainly located on the "slow-low carbohydrate" form. Low affinity, high capacity sites are preferentially located on the "high carbohydrate" form. These results confirm the molecular and functional heterogeneity of rat AFP and suggest that the carbohydrate moiety of the protein may have a role in estrogen-AFP interactions.     

Stimulation of uridine phosphorylation in primary cultures of Xenopus laevis hepatocytes by estradiol-17 beta

The effects of estrogen on the uridine uptake into cells were examined in primary cultures of liver parenchymal cells from Xenopus laevis. The total uptake of [3H]uridine into the estrogen-treated cells and its incorporation into RNA were about 1.5 times higher than the values for control cells. The uptake of [3H]adenosine and its incorporation into RNA were not affected by estrogen. An experiment in which liver parenchymal cells were double labeled with [3H]uridine and [3H]adenosine showed that estrogen elevated the specific radioactivity of the UTP pool 1.4-fold the value found for the control cells, but that of the ATP pool was not altered by estrogen. Short term labeling revealed that estrogen did not significantly alter the rate of the initial uptake of [3H]uridine into the cells, but it did stimulate [3H]uridine phosphorylation about 1.7-fold. Uridine kinase activity measured in cell-free extracts of hepatocytes treated with estrogen had a value 1.6 times that of the control cells. These data indicate that the stimulation of [3H]uridine uptake and phosphorylation in Xenopus laevis hepatocytes in the presence of estrogen is caused by the enhancement of uridine kinase activity.     

Radioimmunoassay of estradiol-17beta in bovine peripheral plasma with and without chromatography.

An assay of estradiol-17beta (E217beta) in bovine peripheral plasma is described. The plasma is incubated with an antiserum to E217beta-BSA and the gamma-globulin fraction precipitated with ammonium sulphate. After extraction with diethyl ether E217beta in the precipitate is estimated by radioimmunoassay using a specific antiserum against E217beta-6-BSA. Plasma concentrations of E217beta during the normal estrous cycle determined by this method and by a method involving Sephadex LH-20 chromatography range from 4 to 23 pg/ml.     

Comparison of the methylation of estradiol-17 beta and of estradiol-17 alpha by dimethyl sulfate

E₂-17β and E₂-17α give substantially similar yields of 3-methyl ether and dimethyl ether when methylated by Brown's procedure.     

Relationship of plasma estradiol-17beta, total estrogen, and progesterone to estrus behavior in mithun (Bos frontalis) cows

The objectives of this study were (1) to establish the characteristics of estrus behavior in mithun cows (n = 12) and (2) to determine the relationships between this behavior and the plasma concentrations of estradiol-17beta (E2), total estrogen, and progesterone. Estrus was detected by visual observations of estrus signs, per recta examination of genitalia and bull parading thrice a day for three consecutive cycles. Among the behavioral signs of estrus, the cow to be mounted by bull (100%) was the best indicator of estrus followed by standing to be mounted (92%). Per rectum examination of genital organs revealed relaxed and open os externa of cervix, turgid uterus, and ovaries having palpable follicles in all animals. The mean (+/-SEM) length of estrus cycle and duration of estrus were recorded to be 21.8 +/- 0.69 days and 12.6 +/- 1.34 h, respectively. Endocrine profiles during the peri-estrus period showed that the mean highest peak concentrations of E2 (27.29 +/- 0.79 pg/ml) and total estrogen (45.69 +/- 2.32 pg/ml) occurred at -3.90 +/- 2.27 and -3.89 +/- 2.26 h prior to the onset of estrus, respectively. Plasma progesterone concentration was basal (0.14 +/- 0.001 ng/ml) during the peri-estrus period. Plasma E2 and total estrogen were found to increase from 6 days before estrus to reach a peak level on the day of estrus and decline thereafter to basal level on day 3 of the cycle. The plasma progesterone concentration was the lowest on the day of estrus showing gradual increase to register a peak level on day 15 of the cycle. Estrus behavior was found to be positively correlated with the maximum peak concentration of E2 (r = 0.89; P < 0.0001) and total estrogen (r = 0.66; P = 0.019) during the peri-estrus period. The mean total estrogen concentration during the peri-estrus period was significantly correlated with estrus behavior (r = 0.60; P = 0.04). The correlations between the estrus behavior and E2:progesterone ratios at 6 days before the onset of estrus (r = 0.92) and on the day of estrus (r = 0.95) was significant. The total estrogen:progesterone ratios at 6 days before the onset of estrus and on the day of estrus were also positively correlated with the estrus behavior (r = 0.86 and 0.88). In conclusion, our results suggest that the maximum peak concentration of E2 and total estrogen and mean level of total estrogen during the peri-estrus period and the E2:progesterone and total estrogen:progesterone ratios on 6 days before the onset of estrus and on the day of estrus are the important factors contributing the behavioral manifestation of estrus in mithun cows.     

Metabolism of estrogens by rabbit blastocysts: formation of estrogen glucosides and preferential conversion of estrone to estradiol-17 beta

When day 6 rabbit blastocysts were cultured (3 embryos/mL) in medium 199 containing 3.68 microM estradiol-17 beta (E2), 40% of E2 was metabolized in 24 h, at a rate of 18 pmol/embryo(b)/h, yielding 4 major metabolite fractions. Two of them were identified to be estrogen glucosides: 17 beta-hydroxyestra-1,3,5(10)-trien-3-yl beta-D-glucopyranoside (E(2)3G) (12 pmol/b/h) and 17-oxoestra-1,3,5(10)-trien-3-yl beta-D-glucopyranoside (E(1)3G) (0.5 pmol/b/h). If the blastocysts were cultured in 3.68 microM E1 medium, 75% of E1 was metabolized in 24 h (34.1 pmol/b/h); most of it appears as E2 (8 pmol/b/h), E(1)3G (16 pmol/b/h), and E(2)3G (6 pmol/b/h). Thus, the 17 beta-hydroxysteroid dehydrogenase activity in the rabbit blastocysts catalyzes mainly in the direction of the E1----E2 conversion, with little or no E2----E1. This may be responsible in part for the faster metabolism of E1 than E2 by the rabbit blastocyst. In comparison with the rat, mouse, and hamster blastocyst, the rabbit embryo shows an additional capability to conjugate large amounts of estrogens into glucosides by steroid glucosyltransferase.     

Culture conditions affect induction of vitellogenin synthesis by estradiol-17 beta in primary cultures of tilapia hepatocytes

In vitro synthesis of vitellogenin (VTG), a female-specific protein, after estradiol-17 beta (E(2)) treatment was compared among different culture conditions using the hepatocytes of tilapia, Oreochromis mossambicus. VTG was measured by enzyme-linked immunosorbent assay. Comparison of Leibovitz's L-15 medium (L-15), Williams' medium E (WE) and Medium 199 (M199), which have been used for hepatocyte cultures in certain teleost fishes, showed that monolayer formation of the hepatocytes on the plate in WE and M199 was faster than in L-15 at the beginning of the culture. Morphological differences in the hepatocytes among the culture media were not evident by 96 h after culture. VTG synthesis in L-15 after E(2) treatment was higher than in WE and M199. A concentration of NaHCO(3) at 5 mM in L-15 resulted in faster monolayer formation of the cells and higher VTG synthesis than at 0 and 23 mM. Primary culture of the tilapia hepatocytes at 28 degrees C showed higher synthesis of VTG than at 23 and 33 degrees C. These results suggest that nutritional requirements are vitally different among species, and there are optimal ranges in the pH and the temperature in cultured hepatocytes.     

Different proliferative responses of periportal and perivenous hepatocytes to EGF.

Stimulation of DNA synthesis by EGF was compared in cultured periportal and perivenous hepatocyte populations. Periportal hepatocytes responded to EGF more sensitive (IC50-values 20 vs 75 ng/ml) and with a higher maximal stimulation (420 vs 290%) than perivenous hepatocytes with respect to both [3H]thymidine incorporation and labeling index. The glutamine synthetase-positive hepatocytes responded much less to EGF than did the perivenous cells in general. The simultaneous presence of insulin increased the sensitivity for EGF predominantly in the periportal hepatocytes. These inherent differences in the growth potential of hepatocytes from different acinar localizations may contribute to different growth patterns across the lobules in normal and regenerating liver.     

Development of a sensitive, direct luminescent enzyme immunoassay for plasma estradiol-17 beta

A rapid, sensitive, precise, chemiluminescent enzyme immunoassay for estradiol-17 beta has been developed and validated. Antibodies were produced in rabbits using estradiol-17 beta-6-(O-carboxymethyl)oxime coupled to bovine serum albumin, purified and immobilized on polystyrene beads (6.4 mm diameter). The same derivative was used to prepare the enzymatic tracer by coupling with horseradish peroxidase. The assay, direct on the serum sample, featured a 4-h binding step at 4 degrees C followed by the chemiluminescent detection using luminol/H2O2. The detection limit was 0.15 pg/tube and the assay was carried out on 20-100 microliter of sample, allowing measurement of estradiol-17 beta in plasma concentrations from 1.5 to 500 pg/ml. The method fulfills all the standard requisites of precision and accuracy and the results agree well with a radioimmunoassay procedure on extracted serum.     

The regulation of rat brain pyruvate kinase by estradiol-17 beta.

The effect of estradiol-17 beta on the activity of pyruvate kinase from rat brain was investigated at initial stages of hormone administration. After estradiol treatment the rise of pyruvate kinase activity was found in the firmly bound synaptosomal fraction, whereas pyruvate kinase activity in soluble and weakly bound fractions has been reduced. The activation of the enzyme was also discovered after glutaraldehyde fixation on synaptosomal membranes. The possible mechanism of extragenomic estradiol action is discussed.     

Metabolic changes in lipids of rat plasma and hepatocytes induced by 17 alpha-ethynylestradiol treatment

Cultured hepatocytes isolated from livers of 17 alpha-ethynylestradiol-treated rats were used to investigate the change of lipid metabolism induced by administration of 17 alpha-ethynylestradiol. Treatment with 17 alpha-ethynylestradiol caused a decrease of rat plasma lipids (free cholesterol, cholesterol ester, triacylglycerol and phosphatidylcholine). No difference in the ability of urea nitrogen synthesis could be demonstrated between cultured hepatocytes isolated from livers of 17 alpha-ethynylestradiol-treated rats and propylene glycol-treated rats (control). Total cholesterol and cholesterol ester contents of cultured hepatocytes isolated from livers of 17 alpha-ethynylestradiol-treated rats were increased in comparison with those of the control. Triacylglycerol content of cultured hepatocytes was not affected by 17 alpha-ethynylestradiol treatment. There was no difference in the composition of lipid content between liver tissues and cultured hepatocytes. These results suggest that hepatocytes isolated from livers maintain the character of livers treated with 17 alpha-ethynylestradiol or livers treated with propylene glycol. Free cholesterol and cholesterol ester synthesis from [14C]acetic acid by cultured hepatocytes isolated from livers of 17 alpha-ethynylestradiol-treated rats were decreased to about 30% of the control. Triacylglycerol and polar lipid (phospholipid) synthesis from [14C]acetic acid were not affected by 17 alpha-ethynylestradiol treatment. Microsomal hydroxymethylglutaryl-CoA reductase activity of rat liver treated with 17 alpha-ethynylestradiol was decreased to about 50% of control. The secretions of free cholesterol, cholesterol ester, triacylglycerol, phosphatidylcholine, apolipoprotein BL and BS by cultured hepatocytes isolated from livers of 17 alpha-ethynylestradiol treated rats were not decreased when compared with the control. Because lipid and apolipoprotein secretions from cultured hepatocytes treated with 17 alpha-ethynylestradiol were not decreased and cholesterol contents of liver tissues and cultured hepatocytes treated with 17 alpha-ethynylestradiol were increased and hepatic microsomal hydroxymethylglutaryl-CoA reductase activity was decreased by 17 alpha-ethynylestradiol treatment, it is suggested that the liver plays an important role in hypolipidemia induced by 17 alpha-ethynylestradiol by increasing the plasma lipid uptake mediated by an increased amount of lipoprotein receptors of liver membranes.