Estrogen-binding Component Research Articles

Heterogeneity and distribution of estrogen binding sites in guinea pig seminal vesicle

Cytosol fractions from the separated epithelium and fibromuscular stroma of the guinea pig seminal vesicle exhibit two types of [ 3H]17β-estradiol binding. High affinity, low capacity, estrogen-specific sites are observed in each component. in addition, secondary binding sites with moderate affinity ( K d = 35 nM) and high capacity (500 fmol/mg cytosolic protein) are also observed. in epithelial cytosol, 10 mM sodium molybdate or 1 mM dithiothreitol enhance [ 3H]17β-estradiol binding to the secondary sites. in stromal cytosol, sodium molybdate is without effect while dithiothreitol completely abolishes binding of [ 3H]17β-estradiol to the secondary sites. Specificity studies indicate that [ 3H]17β-estradiol binding to the secondary sites in stromal cytosol is inhibited by estrogens, but not by androgens. in epithelial cytosol estrogenic specificity is not evident. These results demonstrate the existence of [ 3H]17β-estradiol binding sites in the fibromuscular stroma which are similar to the Type II estrogen binding components present in rat uterus and prostate. The secondary sites in epithelium may in part, represent binding of [ 3H]17β-estradiol to another steroid hormone receptor present in this tissue component. These results also suggest a possible mechanism which may account for the known sensitivity of the seminal vesicle fibromuscular stroma to estrogen stimulation.

Steroid receptor status of focal-nodular hyperplasia of the human liver.

As with normal human liver, both focal nodular hyperplasia (FNH) of the liver and a hepatocellular carcinoma were found to contain low levels of high affinity estrogen-binding components. In low-salt sucrose gradients the estrogen binders sedimented at 4S and 8S. Specificity studies indicated a requirement for estrogenic hormones. Progestin-binding studies using labeled and unlabeled ORG 2058 revealed high levels of specific binding in FNH and normal liver cytosol, whereas only insignificant amounts of ORG 2058 binding was found in the hepatocellular carcinoma. As judged by the high apparent dissociation constant (4-14 X 10(-8) mol/l) of the ORG 2058 - binder complex, the apparent lack of 8S binding in low-salt sucrose gradients and the atypical ligand specificity, the progestin receptor nature of these binding entities is unlikely. In contrast, normal liver, FNH, and the hepatocellular carcinoma were found to contain significant quantities of glucocorticoid receptors.

Mechanisms of estrogen-induced myelotoxicity: Evidence of thymic regulation

Mice exposed to pharmacological levels of steroidal and nonsteroidal estrogens including α-dienestrol, 17β-estradiol, and diethylstilbestrol demonstrate bone marrow hypocellularity, and decreased numbers of pluipotent hemopoietic stem cells. Hormones with little estrogenic activity including testosterone and progesterone failed to induce myelotoxicity as did nonestrogenic metabolites of DES. Myelotoxicity associated with estrogen exposure is regulated by a complex bimodal mechanism. One of these mechanisms is mediated through the thymus since surgical thymectomy abolished the ability of estrogens to suppress CFU priliferation. Furthermore, supernatants of thymic epithelial cells cultured in the presence of estradiol were capable of inhibiting CFU-GM colony formation. Specific myelotoxic events can also be disassociated chemically by testing weakly estrogenic compounds such as zearalanol which shows different sensitivity on cytoxic and antiproliferative events. Myelotoxicity is not mediated indirectly through the ovary or adrenal gland. That the initial events in estrogen-induced myelotoxicity may be mediated through a receptor mechanism was suggested by the ability of antiestrogens to induce antagonism when administered prior to estradiol and the presence of estrogen binding components in lymphoreticular tissues including the thymus and bone marrow. These studies suggest that reduced CFU kinetics observed following estrogen exposure is, in part, due to alterations in regulatory factors produced by thymic epithelial cells in response to a specific estrogen stimulus. Estrogens may also influence bone marrow functions through non-thymic mechanisms at higher dose levels.

Characterization of an estrogen receptor in the turtle testis

Using DNA-cellulose affinity chromatography, an estrogen-binding component having the properties of a classical estrogen receptor was characterized from testicular cytosol of the freshwater turtle, Chrysemys picta. This putative cytoplasmic receptor exhibited high affinity ( K d = 7.0 × 10 −10 M), low capacity (1–4 fmol/mg protein), and steroid binding that was specific for estrogens. It was not present in plasma, muscle, kidney, or lung. A temperature-dependent conversion of turtle testicular estrogen receptor from 4 to 5 S occurred on DNA-cellulose columns, and resembled that in mammalian testis and other target tissues. After a single injection of estrogen at 3 hr, cytoplasmic receptors were depleted with a concomitant increase of nuclear receptors. Identification in turtle testis of an estrogen-binding macromolecule having the physicochemical properties of mammalian estrogen receptors is further evidence that receptors have been widely conserved in many tissue types through vertebrate phylogeny and supports the idea that the testis is an important target of estrogen action.

Characterization of an estrogen receptor in the testis of the urodele amphibian Necturus maculosus.

The exceptionally high rate of aromatization previously demonstrated in the testis of the urodele amphibian Necturus maculosus prompted us to investigate estrogen binding activity in this tissue. When crude cytosolic extracts were incubated with [3H] estradiol prior to DNA-cellulose affinity chromatography or sucrose gradient centrifugation, no estrogen binding was detected. Under these conditions, the presence of an endogenous estrogen-conjugating system in the crude cytosol resulted in extensive conversion of radiolabeled tracer to sulfoconjugates which presumably were poor receptor ligands; however, when this enzyme was first removed from the crude cytosol by fractionation through DNA-cellulose columns, DNA-adhering components could then be labeled with radioactive estradiol. This post-labeling method revealed an estrogen binding component with the physicochemical properties of a classical estrogen receptor: high affinity (Kd=10−10); low capacity (2–5 fmol/mg protein); and affinity for estrogenic steroids only. On sucrose gradients the estrogen receptor complex eluted from DNA-cellulose had a sedimentation coefficient of 4.7–5S. Estrogen receptors having properties similar to the cytosolic form were also demonstrated in nuclear extracts prepared from testes of untreated animals, suggesting that translocation of estrogen synthesized from endogenous precursors is a normal in vivo occurrence in this species. Preliminary measurements indicate that both cytoplasmic and nuclear estrogen receptors may show seasonal changes keyed to the annual reproductive cycle. We reported earlier that estrogen binding activity in the crude cytosol of Necturus testis is located mainly in regions comprised of differentiating or fully differentiated Leydig cells. Further studies of aromatization, the estrogen receptor system, and the estrogen-conjugating enzyme in Necturus testis may provide new insights into the role of intratesticular estrogen in male reproduction.

Acquisition of regulatory mechanisms for gonadotropin receptors and steroidogenesis in the maturing rat testis.

Testicular LH, lactogen, and estrogen receptors and in vitro steroidogenic and cAMP responses were measured in rats between the ages of 1 and 60 days. Testicular LH and lactogen receptors increased gradually with advancing age, although there was no direct correlation between the concentrations of the two receptors. Cytosolic estrogen receptors were present in the testis at 6 days of age, but α-fetoprotein represented the major estrogen-binding component until 20 days of age. The basal and maximally stimulated in vitro production rates of testosterone, pregnenolone, and cAMP per unit weight were more than 10-fold higher in neonatal rats than in 60-dayold animals. These production rates decreased rapidly to a nadir at 15 days and increased again between 15 and 60 days of age. Treatment of 1-day-old rats with a low dose of hCG (10 IU/kg daily) for 3 days caused an increase of 91% (P < 0.01) -in LH receptors per testis. This effect of hCG diminished gradually with advancing age and became insignificant in animals older than 23 days. A single high dose of hCG (600 IU/kg) caused rapid loss of free testicular LH receptors within 12–24 h in animals of all ages. The recovery of LH-binding sites occurred within 2–3 days in 5-day-old animals, but became progressively slower with advancing age and reached 14 days in 60-day-old rats. The in vitro testosterone responses of testes from 5-dayold rats were increased by 2- to 3-fold when measured 3 days after the high dose of hCG. This positive effect of hCG treatment also decreased with age and became insignificant in animals older than 20 days. In adult animals, the same hCG treatment caused complete loss of testosterone responses to gonadotropic stimulation, consistent with the development of steroidogenic lesions in the Leydig cell. These results demonstrate that the production of testosterone, pregnenolone, and cAMP per unit tissue weight is higher in the neonatal testis. Also, hCG has a rapid stimulatory effect upon LH receptors and steroidogenesis in the neonatal testis, but does not cause LH receptor downregulation and steroidogenic lesions at this age. These stimulatory responses to hCG gradually diminish during development and are replaced by the adult-type responses, with sterodogenic lesions and LH receptor down-regulation. The transition from neonatal to adult-type responses which occurs around 15 days of age emphasizes the functional differences between fetal and adult populations of Leydig cells. The development of estrogenmediated inhibitory actions in the testis may contribute to the differences observed between neonatal and adult rat Leydig cells.

Steroid Hormone Receptors in the Rat Mammary Adenocarcinoma Induced by &lt;italic&gt;N&lt;/italic&gt;-Hydroxy-&lt;italic&gt;N&lt;/italic&gt;-2-fluorenylacetamide&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;&lt;xref ref-type="fn" rid="FN3"&gt;3&lt;/xref&gt;&lt;xref ref-type="fn" rid="FN4"&gt;4&lt;/xref&gt;

The steroid hormone receptors in N-2-fluorenylacetamide (2-FAA)- and N-hydroxy-2-FAA-induced mammary adenocarcinomas in outbred Sprague-Dawley rats were analyzed. Both 8S and 4S estrogen-binding components have been detected in cytosols of these tumors following sucrose gradient sedimentation in low salt. Competitive binding analyses of this binder indicated a specificity profile expected of an estrogen receptor. Both androgen and progesterone receptors were also present in the cytosols of these mammary tumors. While the androgen receptor sedimented in the 8S region of the gradient, the progestin binder appeared only as a 4S moiety under similar conditions. The relative concentrations of these receptors (expressed in fmol/mg protein +/- SE) were: 17 beta-estradiol (28.6 +/- 4.1) greater than 5 alpha-dihydrotestosterone (8.5 +/- 2.2) greater than progesterone (5.0 +/- 1.3). The progesterone receptor was increased at least eightfold in the mammary adenocarcinomas from ovariectomized rats that were treated with diethylstilbestrol for 6 days. Binding equilibrium data indicated Ka = 1.2-1.8 X 10(9) M-1 for the above cytoplasmic hormone receptor complexes (Ka, association constant). Although cytosols prepared from lactating mammary gland contained appreciable quantities of glucocorticoid receptor, only trace amounts were found in the mammary adenocarcinoma.

Estrogen receptors in the turtle brain

Estrogen binding activity in the CNS of the freshwater turtle, Chrysemys picta, was investigated using DNA-cellulose affinity chromatography. An estrogen binding component (EBC) with the characteristics of an estrogen receptor species was demonstrated in the brain cytosol extracts from both sexes. This EBC exhibited high affinity ( K d = 10 −10M), low capacity ( n= 0.8 to 6.0fmol/mg cytosol protein) and binding specificity. The bound estradiol-17β which adhered to DNA-cellulose was sensitive to excess synthetic and natural estrogens (DES, E 2, E 1 and E 3) but not to progesterone and androgens (5α-DHT and T). Specific estradiol-17β binding was not detected in plasma or non-target tissues such as lung, kidney and muscle. The topographic distribution of cytoplasmic EBC was similar in males and females, with binding highest in the hypothalamus-preoptic areas (HPOA) followed by the remaining forebrain (RFB). The mid/hindbrain (HB), consisting of the optic lobes, cerebellum and underlying brain stem had significantly lower concentrations of EBC. Monthly data (from May to October) suggest that variations in EBC concentrations occur during the year.

Properties of estradiol binding components in hamster uterine nuclei

Only one estrogen-binding component (Type I) was observed in salt (0.5 M KCl) extracts of proestrous hamster uterine nuclei. In addition to the classical estrogen receptor (Type I), a second binding component (Type II) was detected by [ 3H]estradiol exchange assay performed with hamster uterine nuclear suspensions. Although this Type II binder was not detected in salt extracts, a similar binding component was found in the nuclear debris remaining after salt extraction. The Type II binding component in the nuclear debris did not posess estrogen-binding specificity. Lack of specificity for estrogens, resistance to KCl extraction, and high capacity differentiated this Type II binder from the classical estrogen receptor. Preparation of nuclear fractions in buffer containing glycerol and monothioglycerol resulted in greater recovery of nuclear estrogen receptor (Type I) as compared to buffer lacking these constituents.

Effect of gonadectomy on the ontogeny of estrogen-binding components in rat liver cytosol.

These studies elucidate the ontogeny of two classes of estrogen-binding sites present in rat liver cytosol. The first class of sites is precipitated from whole cytosol fractions by ammonium sulfate (30% saturation) and exhibits characteristics of specific estrogen receptors. Detectable levels of receptors are attained during the third postnatal week. During days 30--40, receptors reach maximum concentration and remain relatively constant thereafter. The second class of sites, detected in whole cytosol fractions, possess a high binding capacity for estrogens and are present in similar amounts in male and female liver before day 34. However, between days 34--40 male levels increase dramatically while female levels remain constant. This sex difference is maintained throughout the duration of the study (160 days). Specific estrogen receptors from immature (26 days) and mature (70--80 days) rat liver have similar characteristics in terms of sedimentation properties in sucrose gradients, ligand binding specificity, and heat and pronase susceptibility. After prepuberal (19--21 days) gonadectomy, levels of receptor in subsequent adult animals of both sexes are increased approximately 40%. No alterations in receptor levels are seen after neonatal (day 1) castration of males. Prepuberal (day 19) gonadectomy does not alter the normal development of sexual differentiation of high capacity estrogen-binding sites. However, after neonatal (day 1) castration male rats, levels of those sites do not undergo sexual differentiation, and neonatally castrated adult males exhibit female levels of high capacity estrogen-binding site. These studies suggest that sexual differentiation of high capacity estrogen-binding sites may be programmed at birth by testicular androgens.

Detection of estrogen receptor-like high affinity components by exposure of cytosols from mouse leydig cell tumor or male rat liver to chaotropic salts

Modifications of steroid-macromolecule interaction induced by so-called chaotropic salts were studied. When the cytosol from one of estrogen-independent mouse Leydig cell tumors was incubated with [ 3H]-estradiol (E 2), an unusual estrogen binding component was identified, with a relatively weak affinity for E 2 ( K d 10 −7 M). This binding could not be inhibited by diethylstilbestrol (DES). However, exposure of this cytosol to 0.4 M NaSCN resulted in the clear demonstration of high affinity binder for E 2 ( K d 10 −9 M). Furthermore, this high affinity binding with E 2 was inhibited by DES in a competitive manner. The quantitation of the binding sites revealed that the binding capacity at low concentrations of [ 3H]-E 2 (less than 10 nM) is higher in the presence of NaSCN than in the absence of NaSCN. Liver cytosol from noncastrated male rat was also found to contain E 2 binding component with a low affinity ( K d 10 −7 M) and a very high concentration of sites. The binding component with high affinity ( K d 10 −9 M) as well as a concomitant loss of the low affinity sites became detectable in 0.4 M NaSCN. Moreover, this association with E 2 was found to be competitively inhibited by DES ( K i 3 × 10 −9 M). The similar changes were elicited by the other chaotropic salt, NaClO 4. These observations suggest that the detection of receptor-like high affinity binder in chaotropic salts may be mediated by two different mechanisms: one is a formation of new high affinity binder, the other is a destruction of the unusual second binder. Furthermore, the easy demonstration of estrogen receptor in chaotropic salt which is not detectable in a conventional buffer system might indicate that careful consideration is required to decide the absence of receptor protein.

Estrogen binding component of mouse Leydig cell tumor: an in vitro conversion from nonreceptor to receptor-like molecule.

The estorgen-binding components in the cytosols prepared from estrogen-dependent (tumor code 22137) and -independent (tumor code 124958) mouse Leydig cell tumors were examined. The estrogen-dependent tumor was found to contain the typical estrogen receptor. The estrogen-independent tumor has a unique estrogen-binding component with low affinity for 17β-estradiol (E2; Kd = 10-7 M) and no affinity for diethylstilbesterol (DES). However, removal of small molecules from the cytosol by dialysis, gel filtration, or ammonium sulfate fractionation resulted in a marked increase in affinity for E2 (Kd = 1O-9-10-10 M) and DES. Similar changes in the dissociation constant and steroid specificity were induced by simply allowing the cytosol to remain at 0 C for more than 18 h or exposing the cytosol to 0.4 M NaSCN for 30 min. These treatments, which induced the tight binding of [3H]E2, made it possible to determine the sedimentation constant in a sucrose gradient. Gel filtration, dialysis, or allowing the cytosol to st...

Estrogen receptor characterization following selective sedimentation separation of estrogen-binding components in immature rat uterine cytosol.

AbstractCharacterization of a specific estrogen receptor (ER) in fetal and early postnatal rat uterine cytosol is complicated by the presence of other high-affinity estrogen-binding components, such as alpha-fetoprotein (AFP). In an attempt to circumvent their influence, we have employed the selective sedimentation of unlabeled cytosol through sucrose gradients, followed by the analysis of [3H]estradiol binding to a pool of fractions comprising the ER region, as well as to individual gradient fractions. As the amount of AFP present in 21-day-old rats is sufficiently low to permit ER characterization by conventional methodology, we have validated the selective sedimentation method by comparing its results with those obtained conventionally. Though conventional gradient analysis revealed only one estrogen-binding component, saturation and binding inhibition analyses indicated the presence of multiple components, identified as AFP and the ER. These conclusions were supported by results from labeling individu...

Resolution of estrogen binding species in hypothalamus and pituitary.

In addition to the classic Type I estrogen receptor, a second estrogen binding species has been reported in rat uterine nuclear material. We have examined nuclear material from hypothalamus and pituitary for the presence of Type II estrogen receptor by the sucrose pad/exchange assay. Extensive (0.05-40.0 nM) saturation analyses were performed on crude chromatin isolated from hypothalami and pituitaries of hyperestrogenized ovariectomized rats. Analysis of data by an adaptation of the graphical method of Rosenthal suggests that there is only a single class of estrogen binding sites in these tissues with a Kd (0.2 nM) close to that reported for Type I receptors in other systems. However, a definitive resolution of the binding component(s) was not possible due to noise in the upper regions of the saturation plot. Therefore, we pooled 7 hypothalamic and 4 pituitary saturation experiments and analyzed the data with LIGAND, a nonlinear curve fitting program. Computer generated curves indicate that the data from both target tissues are approximated most closely by a model describing a single high affinity binding species (Type I) (Kd = 0.17 - 0.38 nM) and a linear or very low affinity nonspecific binding component. Thus, we conclude that there is no evidence for the presence of a second or lower affinity estrogen binding component in hypothalamus or pituitary. The absence of this binding species in these two estrogen target tissues is consistent with the concept that Type II sites may be involved in estrogen's control of tissue hypertrophy and hyperplasia--estrogen responses not found in hypothalamus or pituitary.

Occurrence of estrogen binding components in mouse thymus: A study using fluorescence technique

The occurrence of estrogen binding components was studied in frozen sections from different organs from adult and neonatal female mice. The sections were incubated with a fluorescein-BSA-estradiol-17β complex and studied in a microscope equipped for epi-illumination. A pronounced fluorescence was seen in the reticuloepithelial cells of the thymus while the thymic lymphocytes were negative. Treatment of ovariectomized females with estradiol and progesterone in combination strongly increased fluorescence. The estrogen thymolytic effect is supposed to be due to an interaction between reticuloepithelial cells and thymocytes.

Inhibition of Estrogen Tumorigenesis in the Syrian Golden Hamster Kidney by Antiestrogens&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;&lt;xref ref-type="fn" rid="FN3"&gt;3&lt;/xref&gt;

Both 8S and 4S estrogen-binding components were identified following sucrose gradient analyses in untreated and either diethylstilbestrol (DES)-primed or 17β-estradiol-primed Syrian golden hamster renal cytosols homogenized in the presence of 5% glycerol. The 8S estrogen-binding moiety increased in the untransformed kidney with continued hormone treatment. The concentration of total estrogen receptor in hamster renal cytosols was enhanced from 6.1 ±0.9 fmol/mg protein in untreated hamsters to 20.7±2.5 fmol/mg protein (SEM) in DES-treated hamsters. Binding equilibrium measurements (Ka) for the kidney estrogen binder in DES-primed hamsters was estimated to be 2.4×1010 M−1. The antiestrogens nafoxidine and enclomiphene but not 1-(ρ-2-diethylaminoethoxyphenyl)-1-phenyl-2-ρ-methoxyphenyl ethanol (MER-25) appreciably inhibited the binding activity of the tritiated estradiol, particularly in the 8S region of the gradient, at five hundredfold excess radioinert antiestrogen concentrations. Injection of these antiestrogens for 3 days into previously estrogen-treated hamsters yielded competition profiles in the renal cytosols of these animals consistent with those found after in vitro incubation. Simuitaneous treatment with antiestrogens such as either nafoxidine or enclomiphene and DES completely suppressed the induction of renal carcinoma in the hamster by estrogen. The presence of tumor foci was ascertained by histochemical staining in fresh-frozen sections for esterase activity, i.e., very low levels in tumor foci compared to high levels in adjacent untransformed renal cortex. This observation was consistent with gel electrophoretic data that indicated a marked decline in individual esterase activity in extracts of pure renal tumor compared to normal kidney. In contrast, MER-25 did not inhibit renal tumorigenesis in the hamster in the presence of DES under similar conditions. Therefore, the ability of these antiestrogens to block renal tumorigenesis in the presence of estrogen correlated well with the antiestrogen competition data for the renal estrogen receptor in DES-treated hamsters.

Estrogen receptors in uterine plasma membrane

In order to assess the subcellular distribution of estrogen-binding components in their native state, plasma membrane and other cell fractions were prepared by quantitative methods from uterine cells in the absence of [ 3H]-estradiol-17β (E 2β). Cells isolated from uteri of ovariectomized rats were disrupted with minimum homogenization in buffered isotonic sucrose with CaCl 2 and fractionated using isotonic media throughout. Activities of succinate dehydrogenase and acid phosphatase were concentrated in the mitochondria:lysosome(M + L) fraction. Glucose-6-phosphatase occurred predominantly in the microsome-rich (P) fraction. Alkaline phosphatase and 5'-nucleotidase were found principally in P and crude nuclear (N) fractions, with N also retaining 96% of DNA. Determinations of specific [ 3H]-E 2β binding to cell fractions at equivalent protein levels were conducted by equilibration for 2 h at 4°C. Binding-sites for E 2β were present in N > P > M + L > 105,000 g supernates (S). However, by using alternative homogenization procedures known to elicit lysis, fragmentation, and stripping of cell structures, E 2β binding-sites as well as 5'-nucleotidase, a plasma membrane marker enzyme, both occurred predominantly in S. Using the more conservative cell disruption and fractionation procedures, plasma membrane subfractions (F2) with densities of 1.13–1.16 were partially purified, principally as smooth membrane vesicles and large “ghosts”, by further centrifugation of N in a discontinuous sucrose density gradient. Activity of 5'-nucleotidase in F2 was enriched to 12 times that of the homogenate. Specific binding-sites for E 2β were concentrated in F2 to 23 times homogenate levels. Such binding of E 2β in F2 was saturable, with an association constant of 4.3 × 10 10 M −1. At saturation, E 2β receptors in F2 correspond to 2.0 pmol/mg membrane protein. Ligand specificity of [ 3H]-E 2β binding to F2 was established by negligible competition by 200-fold molar excess of unlabelled estradiol-17α, cortisol, testosterone, or progesterone, whereas E 2β and diethylstilbestrol were effective inhibitors. Specific binding of E 2β to F2 at 4°C was blocked by prior exposure of membranes to trypsin or to 60°C, but remained essentially undiminished by extraction of membranes with either severely hypotonic or high-salt buffers. In controlled experiments, it was found that only 6–9% of [ 3H]-E 2β-labelled cytosol components became associated with F2 during 2 h incubation at 4°C; the bulk of such adsorbed material was readily extracted by high-salt buffers. Thus, enrichment of F2 in E 2β-binding activity cannot be attributed to gross entrapment or adsorption of cytosolic components. These data indicate that high-affinity membrane binding-sites with specificity for E 2β must be further considered in investigations of the uterine cell recognition of and response to the hormone.

Estrogen-Independent Nuclear Binding of Receptor Protein of Rat Uterine Cytosol by Removal of Low Molecular Weight Inhibitor*

A cell-free system prepared from the ovariectomized rat uterus was used to study the uptake of the cytoplasmic estrogen receptor (RE) by isolated nuclei. At 0–4 C, dialysis of cytosol containing [3H]estradiol (E2)-RE complexes resulted in a 3- to 6-fold increase in nuclear binding of [3H]E2-RE in comparison with nondialyzed cytosol. Removal of the low molecular weight materials by Sephadex G-25 chromatography also enhanced binding of [3H]E22-RE complexes to nuclei. Incubation of nuclei at 0 C with cytosol dialyzed in the absence of estrogen revealed the appearance of the estrogen-binding component in nuclei which showed high affinity for EE (Kd = 6 × 10-10 M at 0 C). This nuclear estrogen-binding component was not occupied with estrogen, since a slightly higher amount of binding with [3H]EE was obtained at 0 than at 37 C. The effects of dialysis on the sedimentation constants were also studied. Dialysis of [3H]E2-RE complexes in cytosol at 0–4 C caused a change of the sedimentation constant from 4.3S to 5...

Estradiol receptors in the pituitary and anterior hypothalamus of the rat: Measurement by agar gel electrophoresis

Agar gel electrophoresis has been applied to the measurement of the high-affinity saturable estrogen binding component in the cytosol of rat pituitary and anterior hypothalamus. Under the conditions employed, the number of available binding sites can be determined in small samples with good precision and accuracy. The total number of estrogen binding sites in the anterior hypothalamus of 28-day-old females was measurably (⋍15%) greater than that in males. A significant difference in the pituitary could not be demonstrated. The total number of binding sites in the pituitary was twice that in the anterior hypothalamus in both males and females.

Inhibition of embryonic development in Rana pipiens by 2-methyl-3-ethyl-4-phenyl-4-cyclohexene carboxylic acid (ORF 3858).

2-methyl-3-ethyl-4-phenyl-4-cyclohexene carboxylic acid (ORF 3858) was tested in Rana pipiens embryos to determine its effects on embryonic development and whether such effects are related to its estrogenicity. Development was blocked at cleavage gastrulation neurulation or hatching stages. Sensitivity to the compound increased as development proceeded. There was no correlation between the rate of embryonic uptake of the compound and its ability to block development. Receptor studies showed that uptake of estradiol and the estrogenic metabolites of ORF 3858 by frog embryonic cells did not involve specific high-affinity estrogen-binding components. Neither ORF 3858 nor estrogens modified embryonic nucleic acid synthesis. The compound appears to be unaltered by embryonic metabolism and seems to act as a zygolytic agent.