The uptake of radioactivity by the uterus of the rabbit following the sytemic administration of (3H) estradiol-17beta and (3H) estrone and the identification and subcellular localization of radiometabolites in the uterus

In an effort to determine the relevance of the uterine oxido-reduction of estrogens to their action in the rabbit uterus, the uterine uptake of radioactivity administered subcutaneously as [³h] estradiol-17β or [³H]estrone and the subcellular distribution of radio-metabolites in the uterine tissue were studied. The animals were killed 20 min, 1, 3 and 9 hr after the administration of 0.1 μg tritiated steroid and the relative proportions of radioactive estradiol-17β and estrone in plasma and in ‘cytosol’, ‘mitochondrial/microsomal’ and ‘nuclear’ fractions of the uterine homogenates were studied. Despite the presence of a high proportion of estrone in chloroform extract of plasma, very little was found in the fractions from uterine tissue irrespective of the steroid administered. Highest levels of uterine estrone were found in the ‘mitochondrial/microsomal’ preparation. There was no apparent difference in the pattern of uptake of radioactivity administered as [³H] estradiol-17β or [³H] estrone. The presence of high levels of 17β-hydroxysteroid dehydrogenase activity in the rabbit uterus may be responsible for the apparent difference between these results and those of similar experiments using the rat.     

A comparison of the in vitro metabolism of estrone,estradiol-17β and estrone-3-sulfate by the livers of young virgin female,pregnant and female fetal guinea-pigs

The metabolism, $\text{in vitro}$, of [³H]-estrone, [³H]-estradiol-17β and [³h]-estrone-3-sulfate by the livers of pregnant, young virgin female and female fetus guinea-pigs has been compared using 900 g supernatants and microsomes. The ability of the guinea-pig livers to synthesize polyhydroxylated estrogens has been found to be small. The major metabolites isolated were unconjugated estrone and estradiol-17β or their glucuronides. The percentage of sulfates was lower after incubations with [³H]-estrone than with [³H]-estradiol-17β. A kinetic study with microsomes has shown a direct conversion of estrone-sulfate to estradiol sulfate. Fetal microsomes have been found to exhibit a more active hydrogenation of estrone to estradiol-17β than microsomes from young female or pregnant animals.     

Steroidal control of rat uterine 17β-hydroxysteroid dehydrogenase activity

The interconversion of estradiol-17β and estrone in the rat uterus is due to the action of 17β-hydroxysteroid dehydrogenase. Whole uteri or 800 × g supernatant fractions of the uteri were incubated in the presence of [³H] estradiol-17β and NAD at 37°C for 3 h or 1 h, respectively. In the mature rat uterus the oxidation of estradiol-17β and estrone was dependent on the stage of the estrous cycle, suggesting hormonal control. The 17β-hydroxysteroid dehydrogenase activity was highest at estrus (200 fmol estrone) and lowest at diestrus (80 fmol estrone). An enhancement of activity occurred when adult rats at each stage of the estrous cycle were administered estradiol-17β, while progesterone administration at each stage resulted in decreased enzyme activity. The uterine 17β-hydroxysteroid dehydrogenase activity of estradiol-17β treated ovariectomized rats was time and dose dependent but decreased when progesterone was administered with or without estradiol-17β administration. These results suggest that estradiol-17β caused an increase in enzyme activity that was inhibitable by progesterone in the rat uterus. The increased 17β -hydroxysteroid dehydrogenase activity may reflect a specific response of the rat uterus to estradiol-17β.     

Brief Report: Differential oxidations of estradiol-17β by the chimpanzee in vivo

The metabolism of estradiol-17β is primarily an oxidative process at either carbon-2 or carbon-16 in the human. The objective of this study was to determine the relative importance of these two oxygenation pathways in the chimpanzee. The rate of oxidation of estradiol-17β at each position was determined by measuring the release of tritium into body water from carbon-2 or carbon-16. [2-³H]-Estradiol-17β or [16-³H]-estradiol-17β was injected intravenously into three adult male chimpanzees, and blood samples were obtained at several time intervals between 1 and 48 hr. The blood was lyophilized, and the release of tritium from the specifically labeled estrogens into the body fluid pool was determined. The release of tritium from the 16α-position was very low and did not exceed 3% in any animal. The release of tritium from the carbon-2 was much faster, amounting to 29%, 34%, and 35%, respectively, by 24 hr. The ratio of tritium released from carbon-2:carbon-16 was 5.0, 13.2 and 16.9, respectively, at 24 hr after injection of the specifically labeled estradiol-17β. These results demonstrate clearly that the major pathway for oxidative metabolism of estradiol-17β in the chimpanzee is via oxygenation at carbon-2, with the formation of catechol estrogens, as in the human.     

Metabolites of estradiol-17β in guinea pig uterus late in pregnancy

The metabolism of estradiol-17β by the guinea pig uterus late in pregnancy was studied $\text{in vivo}$ and $\text{in vitro}$.Whole uteri were examined for estrogen metabolites one hour following an intravenous injection of [³H]-estradiol-17β or uterine sections were examined after incubation for one hour at 37°C in medium containing [³H]-estradiol-17β.In both instances uterine tissue metabolized estradiol-17g to five products: estrone, estrone-3-sulfate, 17β-estradiol-3-sulfate, estrone-3-glucuronide and 17β-estradiol-3-glucuronide. Of the total radioactive products 11 – 43% were glucuronides, 17 – 26% were sulfates and 4 – 17% was estrone. These results indicate that the guinea pig uterus actively transforms estradiol-17β into glucuronides and sulfates late in pregnancy.     

Antiestrogenic and antifertility actions of anordrin (2α,17ga-diethynyl-A-nor-5α-androstane-2β,17β-diol 2,17-dipropionate)

Anordrin, administered in a single s.c. dose of 62.5 μg in sesame oil, stimulated sustained uterine growth (wet weight) when measured at 24 and 72 hr, but total soluble protein and total DNA per uterus was not increased. By comparison, 3 μg of estradiol-17β under the same conditions significantly increased all three parameters of uterine growth. Both of the above steroid treatments significantly increased nuclear estrogen receptor content of the uterus, but only the estradi-ol-17β treatment resulted in significantly elevated cytosol receptor content per uterus. Anordrin binds to the 8S estrogen receptor with an affinity of about 2 × 10⁵ M^-1 as determined by competition with [³H]estradiol-17β. The abortifacient activity of Anordrin when given orally (8 mg/kg b.w.) to mice on the 7th day of pregnancy was almost completely blocked by simultaneous oral administration of estradiol-17β (0.8 mg/kg b.w.). It is concluded that the actions of Anordrin on the uterus can be attributed to its antiestrogenic activities.     

Steroid estrogen conjugates in hens' urine II. identifications of some minor conversion products of intramuscularly injected [4-14c]estrone

[4-¹⁴C]Estrone was injected intramuscularly into two mature laying Rhode Island Red hens. Radioactive steroids and steroid conjugates recovered from the urine on Amberlite XAD-2 columns were fractionated on columns (100 cm) of DEAE-Sephadex A-25 by NaCl gradients. The presences of the following were confirmed, the figures in brackets indicating average proportions as per cent of total radioactivity recovered after Sephadex column chromatography: -the 3-β-glucuronides of estrone (10. 9) and of estradiol-17α plus estradiol-17β(9.8); the 17-β-glucuronides of estradiol-17α plus estradiol-17β (2.1); the 3-sulfates of estrone (14. 5) and of estradiol-17α plus estradiol-17β (27. 4); and the disulfates of estradiol-17α plus estradiol-17β (2. 3). The following additional conjugates were identified:-a β-glucuronide of 16-epiestriol (0.2) and a β-glucuronide of 16-ketoestradiol-17β (0. 2); the 3-sulfates of 16-epiestriol (1. 4), of 17-epiestriol (0. 9), of 16, 17-epiestriol (0. 7), of 16-keto-estradiol-17β (1. 1), and of 2-methoxyestrone (0. 7). Some evidence was obtained for the presence of 16, 17-epoxy-estratrienol-3-sulfate (1.9).     

Estrogen mercapturic acids in the adult male rat

N-Acetylcysteine derivatives of catechol estrogens have been isolated from the urine of adult male hooded rats with ligated bile ducts, following injection of [4-¹⁴C]2-hydroxyestradiol-17β and of [4-¹⁴C]estradiol-17β-By application of double isotope methods previously described, it was shown that 2-hydroxyestradiol-17β was converted into mercapturic acids in a yield of 6–8%, confirming two previous experiments with bile duct cannulated rats, and that estradiol-17β was converted into mercapturic acids to the extent of 3–6%. Since these figures are small, and since it has been shown that in two women estrogen mercapturic acids were not formed, it appears that this class of compound will not provide an answer to the problem of unidentified water-soluble metabolites of the estrogens.     

Antagonism of the actions of estrogens,androgens and progesterone by anordrin (2α,17α-diethynyl-A-nor-5α-androstane-2β, 17β-diol dipropionate)

Anordrin, an antifertility agent that is an antiestrogen with weak estrogenic activity, has been studied to further characterize its hormonal activities. A dose of 2.0 μg/mouse·day for 7 days did not increase the uterine content of protein, but it did inhibit to a small extent the effect of administered estradiol-17β on uterine protein content and more significantly the effect of estradiol-17β on the uterine content of progesterone receptors. Anordrin also decreased serum corticosteroid-binding globulin levels. Administration of an average daily dose of 160 μg/day of anordrin to intact male mice had no effect on weights of kidney, testis, or seminal vesicle after 10 days, but seminal vesicle weight was significantly decreased after 30 days at a slightly lower dose. Similarly, anordrin inhibited the increase in seminal vesicle weight induced by testosterone propionate treatment of castrated mice. In female mice anordrin failed to maintain deciduomata and blocked the ability of progesterone (2.0 mg/mouse·day) to do so. However, anordrin did not compete with the androgen [³H]R1881 for binding in kidney cytosol or with the progestin [³H]R5020 for uterine receptor sites. Anordrin also did not compete with [³H]corticosterone for binding to serum proteins.     

Decrease of Hormone Binding Capacity of Estrogen Receptor by Calcium

Abstract

We have adressed the question as to whether calcium may modify the [³H]estradiol ([³H]E₂) binding properties of the estrogen receptor (ER). A human recombinant full length ER (yER) expressed in yeast was used to limit the potential interference of ER-associated proteins and proteases present in the target tissues.

Ca⁺⁺ (0.1–10 mM) always produced an important loss of [³H]E₂ binding capacity without any effect on the hormone binding affinity of residual receptors. This loss was reflected in a decrease of immunoreactivity for monoclonal antibodies raised against the hormone binding domain. An ER recombinant expressing solely this domain confirmed that the ion operated at this level. Binding of [¹²⁵I]Z-17 α-(2-iodovinyl)-11β-chloromethyl estradiol-17β (an compound with very high selectivity for ER) as well as [¹²⁵I]tamoxifen aziridine were similarly affected. Size-exclusion chromatography failed to reveal the emergence of any ER isoforms of low molecular weight rejecting the hypothesis of a Ca⁺⁺- induced proteolysis. In agreement with this conclusion, EDTA reversed the loss of [³H]E₂ binding capacity. Phosphoamino acids (PY, PT and PS) partly antagonized the effect of Ca⁺⁺ suggesting its interaction with phosphoamino acid residues. Worthy of note, the effect of Ca⁺⁺ appeared more marked when assessed by DCC than HAP assay. The phosphocalcic nature of the HAP matrix may explain this phenomenon which was observed with cytosolic ER from various origins.     

Steroidal and growth factor regulation of [3H]thymidine incorporation by cultured endosalpingeal cells of the bovine oviduct

Summary Cultured cells from the bovine endosalpinx were used to evaluate effects of estradiol-17β, progesterone, epidermal growth factor, and insulinlike growth factors I and II on [³H]thymidine incorporation. Cells were treated with hormones and growth factors when approximately 50% confluent. After 24 h, DNA synthesis was quantified by pulsing cells with [³H]thymidine for 12 h and determining uptake into DNA. Cells prepared by mechanical dispersal incorporated more [³H]thymidine than cells dispersed with collagenase. However, hormonal responses were the same for both types of cells. As compared to plastic, cells on a Matrigel substratum exhibited lower incorporation of [³H]thymidine and were unresponsive to hormones. Estradiol-17β increased [³H]thymidine incorporation slightly at 10⁻¹⁰ mol/liter and higher. Epidermal growth factor, insulinlike growth factor-I, and insulinlike growth factor-II also stimulated [³H]thymidine incorporation. Effects of insulinlike growth factor-I were greater for cells treated with estradiol-17β. In the absence of estradiol, progesterone inhibited [³H]thymidine incorporation at 1, 10, and 100 ng/ml. When estradiol-17β was present, progesterone stimulated [³H]thymidine incorporation at 1 ng/ml and reduced incorporation at 100 ng/ml. In conclusion, [³H]thymidine incorporation by cultured oviductal endosalpingeal cells can be regulated by ovarian steroids and growth factors. These molecules may represent signals through which the ovary, embryo, and oviduct regulate oviductal growth. Work conducted while on a sabbatical leave supported by the Deutsche Forschungsgemeinschaft.     

METABOLIC RELATIONSHIPS BETWEEN MYELIN SUBFRACTIONS: ENTRY OF PROTEINS 1

Abstract— Partially purified myelin from brains of 17-day-old rats was separated into 4 subfractions on a discontinuous sucrose gradient by virtue of heterogeneity in density and particle size. The protein composition of each subfraction was determined by densitometry following separation of proteins on polyacrylamide gels in buffers containing sodium dodecyl sulphate. The major proteins studied included two basic proteins, proteolipid protein, the major high molecular weight protein (W) and a group of high molecular weight proteins. The percentage of high molecular weight proteins decreased sequentially from fraction D to A, that of the W protein remained constant, while relative amounts of the two basic proteins increased. Proteolipid protein concentration also increased as a percentage of the total protein from fraction D to B, but the uppermost fraction. A, had a markedly lower amount than fraction B. At 1 h after intracranial injection of [³H]leucine, the specific radioactivity of the basic and proteolipid proteins decreased from fraction D to B, with proteolipid protein in fraction A again anomalous (specific radioactivity higher than expected). These results are consistent with (but do not prove) a precursor-product relationship for individual proteins from denser to lighter subfractions, with the exception of myelin subfraction A. Experiments involving time staggered injections of a [¹⁴C] and later a [³H] labelled amino acid gave data which demonstrated that the W and basic proteins were added simultaneously (or with delays of much less than 20 min) to all of the subfractions, while proteolipid protein was added sequentially, from lower to upper fractions on the gradient. This double isotope technique also confirmed our previous observations that proteolipid protein shows a lag in entry into myelin compared to basic protein.     

METABOLIC RELATIONSHIPS BETWEEN MYELIN SUBFRACTIONS: ENTRY OF GALACTOLIPIDS AND PHOSPHOLIPIDS 1

Abstract— Partially purified myelin from the brains of 17-day-old rats was separated into 4 subfractions on a three-step sucrose gradient by virtue of heterogeneity in density and particle size. Precursor-product relationships between different membrane fractions were investigated by determining the specific radioactivity of individual lipids in each subcellular fraction 15 min after intracranial injection of an appropriate precursor. Rats were injected with [2-³H]glycerol. myelin subfractions prepared, and individual lipids separated by TLC. For choline and ethanolamine phospholipids, specific radioactivity was highest in the densest fraction (D), intermediate in the next densest fraction (C), and lowest in the lighter fractions (B and A). Similar results were observed for cerebroside and sulphatide when [³H]galactose was the precursor. These data are consistent with (but do not prove) a precursor-product relationship for individual lipids from the densest to the lightest subfraction. Another experimental design involving time staggered injections of [³H] and [¹⁴C] precursors was developed which enables a more definitive result with regard to precursor-product relationships to be obtained. A precursor-product relationship between a given lipid in a dense myelin membrane fraction, and the same lipid in a lighter subfraction, would be indicated by a change in isotope ratio. If there is no precursor-product relationship. Ihe isotope ratio should be constant. Such experiments were done with [³H] and [¹⁴C]glycerol. The data indicated that phosphatidyl ethanolamine and its plasmalogen analog were added first to the densest subfraction and then in turn to the lighter subfractions. In contrast, phosphatidyl choline and its plasmalogen analog were added “simultaneously” (i.e. with delays of much less than 15min) to each of the subfractions. Similar experiments with [³H] and [¹⁴C]galactose showed that cerebroside, sulphatide and galactosyl diglyceride also entered the subfractions simultaneously rather than in sequential order. Thus the assembly of the myelin sheath involves an obligate order of addition of certain lipids. while other lipids are probably added in a random order.     

Aromatization of testosterone in large and small bovine luteal cells. Conflicting results between radioimmunoassay and cocrystallization data

The ability of isolated large or small bovine luteal cells to synthesize estradiol-17β was tested by incubations in the absence or in the presence of exogenous testosterone.Using a specific radioimmunoassay, no synthesis of estradiol-17β could be detected in the small or large luteal cells after incubation in the absence of testosterone. However, after incubation in the presence of exogenous unlabelled testosterone, radioimmunoassay data suggested the existence in the large but not the small luteal cells of synthesis of estradiol-17β. However, the results obtained by measuring the conversion of ³H-testosterone to ³H-estradiol-17β by cocrystallization with unlabelled estradiol-17β failed to confirm the presence of aromatase in the large cells. These data indicate that aromatization in large and small bovine luteal cells is probably negligible. Moreover, they cast serious doubt on studies of aromatization in luteal tissue based on radioimmunoassay data only.     

The influence of conjugated estrogens in radioimmunoassays using different antibodies against estradiol-17β

Estradiol-17β 17-D-glucoronide and estrone-3-sulphate were found to give minor crossreaction in radio-immunoassay systems for estradiol-17β using two different types of antibodies. The antibodies used were prepared in sheep against estradiol-17β 17-succinyl-bovine serum albumin and 6-ketoestradiol-17β 6-(0-carboxymethyl)oxime-bovine serum albumin. Only minute quantities of conjugated estrogens were extracted from plasma by diethyl ether (<0.5 %). These two factors limit the interference of conjugated estrogens.     

Conversion,in vitro,of (7n-3H) testosterone to estrone and estradiol-17β and their 3-sulfäte conjugate by the guinea-pig placenta

Different cellular fractions of guinea-pig placenta were incubated in the presence of (7n-³H) testosterone. Microsomal aromatization of ³H-testosterone into estrone and estradiol-17β was demonstrated in the presence of NADPH. The predominance of estrone after incubation with 17β-hydroxylated precursors, (7n-³H) testosterone and (6,7-³H) estradiol-17β, indicate that there is a microsomal 17β-hydroxysterold dehydrogenase activity. In this report, cytosolic sulfurylation of estrogens is demonstrated. This latter activity represents a quite original characteristic of the placental metabolism of estrogens in guinea-pigs. In contrast with the human placenta where there is considerable sulfatase activity, the guinea-pig placenta can sulfurylate estrogens.     

Non-random incorporation of 5-bromodeoxyuridine in rat cell DNA

Secondary cultures of rat embryo cells were exposed for 24 hrs. to 10-⁷M [³H] thymidine (TdR) or 10^?7M [³H]5-bromodeoxyuridine (BrdU) in order to localize and compare the distribution of the isotopes in DNA. DNA was extracted, sheared, and centrifuged to equilibrium through neutral and alkaline CsCl density gradients. The DNA band from each gradient type was separated into a “heavy” and “light” fraction, and DNA-DNA reassociation hybridizations were performed on each sample. Renaturation profiles revealed that each fractionated DNA sample was representative of the complete rat cell genome, except for the “light” [³H]BrdU-DNA prepared by centrifugation through alkaline CsCl gradients. This fraction was predominantly depleted of labeled late repetitive and intermediate sequences. Uncentrifuged rat DNA was sequentially fractionated during reassociation into rapidly, intermediate, and slowly reassociating sequences by hydroxyapatite chromatography. Relative specific activities of each component revealed a non-uniform distribution of [³H]BrdU moieties as compared to [³H]TdR. These results suggest a nonrandom incorporation of 10^?7M BrdU into rat cell DNA sequences.     

Isolation of histidyl peptides of the steroid-binding site of human placental estradiol 17β-dehydrogenase

Human placental estradiol 17β-dehydrogenase (E.C. 1.1.1.62) was inactivated at pH 6.3 by 3-bromo[2′-¹⁴C]acetoxy-1,3,5(10)estratrien-17-one, a known substrate. The affinity-alkylated enzyme was then hydrolyzed by trypsin. Radioactive peptides were initially isolated by gel filtration and identified according to which residue was alkylated. Tryptic peptides containing radioactive 3-carboxymethylhistidyl residues were further purified by cation-exchange chromatography. The population of these peptides varied, depending upon the conditions of enzyme inactivation. With 60 μM 3-bromo[2′-¹⁴C]acetoxy-1,3,5(10)estratrien-17-one four major peptides (a,b,c,d) each containing radioactive 3-carboxymethylhistidine, were eluted from the cation-exchange column. The alkylation of all of these peptides was completely suppressed when the enzyme was inactivated in the presence of excess estradiol-17β. The presence of equimolar NADPH during incubation greatly enhanced the alkylation of all four peptides. In the presence of NADPH, estradiol-17β most significantly decreased the formation of peptide d. Peptide d was the only peptide identified when the concentration of the alkylating steroid was lowered to 6 βM, a value approaching the Km. These observations indicate that peptide d is a histidyl-bearing peptide from the steroid-binding site which proximates the steroid A-ring. They further suggest that with the affinity labeling steroid at higher concentrations other nonspecific, hydrophobic sites on the enzyme are occupied and labeled.     

Identifications of radioactive steroid estrogen conjugates in blood plasma of laying hens after intramuscular injection of (4--14C)-estrone.

[4--14C] Estrone was injected intramuscularly into six laying hens. Fifty minutes later the hens were exsanguinated. The plasmas were examined for conjugates of radioactive phenolic steroids by recovery on columns of Amberlite XAD-2 or by extraction with tetrahydrofuran followed by chromatography on a column of DEAE-Sephadex A-25 in a gradient of NaCl. The biggest Sephadex chromatographic fraction (50,4% of total) contained about 42% of its radioactivity as estradiol-17alpha-3-sulfate and 18% as estradiol-17beta-3-sulfate and the remaining 40% was identified tentatively as estradiol-17alpha-17-sulfate plus a small proportion of estradiol-17beta-17-sulfate. The second biggest Sephadex chromatographic fraction (12.7% of total) was a mixture of conjugates not further identified. Minor fractions identified comprised estrone-beta-glucuronide (2.8%), estradiol-17alpha-3-beta-glucuronide (2.8%), estradiol-17beta-3-beta-glucuronide (2.3%) and estrone sulfate (6.0%). Evidence was obtained for the presence of small proportions of estradiol-17alpha disulfate and estradiol-17beta disulfate.     

Boron estrogens: Synthesis,biochemical and biological testing of estrone and estradiol-17β 3-carboranylmethyl ethers

Synthesis, biochemical and biological testing of the first carborane derivatives of estrogens are described. Estrone 3-carboranylmethyl ether was synthesized in two steps from estrone. Reduction of estrone 3-carboranylmethyl ether with sodium borohydride provided estradiol-17β 3-carboranylmethyl ether. Enzyme kinetic measurements showed that estrone 3-carboranylmethyl ether is a substrate for human placental 17β-hydroxy-steroid dehydrogenase with Km = 5×10^?6M, and Vmax = 0.016 μmol min^?1 μg^?1. The relative affinity constant of estradiol-17β 3-carboranylmethyl ether for rat uterine estrogen receptor was 0.5 (compared with a value of 100 for estradiol-17β). Consistent with its low affinity for estrogen receptor, the dose-dependent uterotropic response to estradiol-17β 3-carboranylmethyl ether in castrated female rats was one sixtieth that of estradiol-17β. None of the tested rats had a toxic reaction to estradiol-17β 3-carboranylmethyl ether. These results demonstrate that exceptionally stable carborane derivatives of estrogens can be synthesized with preservation of their biochemical and biological properties. Boron-containing estrogens may be useful for thermal neutron capture therapy of cancers with estrogen receptors to concentrate boron in the cell nucleus.