Androgen-binding proteins in rat epididymis: properties of a cytoplasmic receptor for androgen similar to the androgen receptor in ventral prostate and different from androgen-binding protein (ABP)

The cytoplasmic receptor (CR) in rat epididymal 105,000 $\text{g}$ supernatant was separated from the androgen-binding protein (ABP) by gel electrophoresis following labeling with [1,2,6,7-³H]-testosterone in vivo. ABP disappeared from epididymal supernatants after castration 01 hypophysectomy, while CR remained unchanged. CR was evenly distributed between caput and cauda, while much more ABP was present in caput. Properties of CR in epididymis and prostate were similar and distinctly different from ABP. Binding to CR was destroyed by charcoal treatment (1 mg/mg protein) of supernatant at 0 °C for 6 h, heating at 50 °C for 30 min, or exposure to the sulfhydryl blocking reagent, $\text{p}$-chloromercuriphenylsulfonate (1 mM) at 25 °C for 30 min, while binding to ABP was unaffected. The isoelectric pH of CR (5.8) was higher than that of ABP (4.6). Dissociation of radioactive 5α-dihydrotestosterone (DHT) from CR and nuclear receptors was extremely slow (half-time at 0 °C >2 days), while dissociation from ABP was rapid (half-time at 0 °C ~ 6 min). Cyproterone acetate (250 mg/100g body weight) inhibited binding to CR both in epididymis and ventral prostate but did not affect binding to ABP. Nuclear uptake was inhibited by cyproterone to the same extent as binding to CR, indicating that nuclear uptake and binding are dependent on CR and independent of ABP. The time-course of uptake and binding in epididymal supernatant and nuclear fractions was essentially the same 1 day after bilateral castration when both CR and ABP were present or 8 days after castration when CR alone was present. It is concluded that the cytoplasmic receptor for androgen in rat epididymis has properties very similar to the androgen receptor in ventral prostate but different from ABP.     

High-affinity binding of the antiestrogen [3H]tamoxifen to the 8S estradiol receptor

The interaction of tamoxifen (ICI 46,474), a synthetic antiestrogen, with uterine cytosol proteins of immature calf and rat has been studied directly using the tritiated compound labeled with a high specific activity. The binding complexes were measured by the dextrancoated charcoal, protamine sulfate and hydroxyapatite assays. Scatchard plots revealed a single class of high-affinity (KD congruent to 1.7 nM) binding sites, with a binding capacity similar to that of estradiol. Competitive experiments showed the same binding specificity for estrogens and antiestrogens. Sucrose gradient analysis revealed an 8S binding protein which could be partially proteolysed by trypsin into a 4S binding protein. Kinetic studies showed that the association rate of tamoxifen was 5 times lower than that of estradiol and reacted according to a second order kinetics. The first-order kinetics of dissociation was considerably higher than that of estradiol, giving a half-dissociation time of 20--40 min at 0--2 degrees C. In some cases tamoxifen displayed two slopes of dissociation, but the proportion of the slow-dissociating complex was always inferior to that found with estradiol. In contrast to estradiol, the kinetic constants ratio (k-/k+) gave a calculated dissociation constant, similar to that determined in equilibrium conditions (KD), agreeing with a simple reactional scheme. We conclude that the antiestrogen tamoxifen binds directly to the 8S cytosol receptor for estrogens and not to another receptor for the antagonists. In contrast to estradiol, the antagonist is rapidly dissociated from the receptor sites and is unable to protect them against thermal inactivation. The affinity of tamoxifen for its receptor sites as determined directly is surprisingly high when compared to its affinity evaluated indirectly by competitive experiments. It is then suggested that the two ligands either bind on two different sites of the same protein or induce a different conformational change of the same binding site.     

Further characterization of the androgen receptor in rat testis.

Immature rat testes contain a specific binding protein for testosterone (T) and 5alpha-dihyrotestosterone (DHT) with physico-chemical properties similar to the cytoplasmic androgen receptors in the epididymis and ventral prostate but different from the testicular androgen-binding protein (ABP). Like the androgen receptors in the prostate and epididymis, it has a sedimentation coefficient of about 7 S at low ionic strength, is eluted in or close to the void volume on Sephadex G-200 gel filtration (Stokes radius greater than 80 A), has an isoelectric point of about 5.6-6.0 (mean) 5.8 and a relative mobility (Rf) of 0.4 in 3.25% acrylamide gels. Following the injection of 3H-labeled testosterone, T and DHT are bound selectively by the receptor. Relatively more [3H]T than [3H]DHT is present in bound and free fractions as well as in total testicular 105,000 g supernatant. Similar results are obtained from testicular incubations with equimolar amounts of [3H]T and [3H]DHT at 0 degrees C in vitro. Saturation of receptor sites is achieved by incubation of testis supernatants with increasing amounts of [3H]T at 0 degrees C. The number of available binding sites following post-hypophysectomy regression is estimated to be about 9 fmoles/mg protein, and the apparent equilibrium constant of dissociation is 7 X 10(-10) M. The temperature stability and sulfhydryl dependence of the testicular androgen receptor are similar to androgen receptors in other organs. Binding is destroyed by heating the supernatants at 50 degrees C for 30 min and by exposure to p-chloromercuriphenylsulfonate (1 mM) at 0 degrees C for 60 min. Furthermore, like other androgen receptors, the half-time of dissociation of testicular androgen-receptor complexes at 0 degrees C is extremely slow (t1/2 greater than 35 h). Separation of seminiferous tubules from interstitial tissue showed that a major portion of these receptors were localized within the seminiferous tubules.     

Glucocorticoid receptor in human embryo fibroblasts. I. Kinetic and physicochemical properties.

The kinetics of dexamethasone binding to L 809 E cell line cytosol have been investigated by means of the protamine sulfate precipitation assay. The KDeq for dexamethasone was 1.1--3.3 nM. Binding was specific for glucocorticoids. The mean association rate constant (k+1) was 8.5 x 10(5) M-1 x min-1 and the dissociation rate constant was 4.6 x 10(-5) min-1 at 0 degrees C. The concentration of binding sites was 0.3 pmol/mg of cytosol protein. Binding kinetics were compatible with a model of positive cooperativity. The receptor sedimented at 7.5--9 S in glycerol gradients. By a combination of calibrated ultracentrifugation and polyacrylamide gel electrophoresis, a Stokes radius of 8.5 nm, a molecular weight of 268 000 daltons and a frictional ratio of 1.8 were determined in low ionic strength conditions. When the cells were incubated with 10 nM [3H]dexamethasone for 1 h, a more than 90% depletion of cytosol receptor and an equivalent accumulation of nuclear dexamethasone--receptor complexes was observed.     

Characterization of a cytoplasmic androgen receptor in the ram testis

An androgen receptor has been characterized in the cytosol fraction of testes from hypophysectomized adult rams after in vitro labelling with [3H]testosterone. It can be distinguished from the testicular androgen-binding protein (ABP) and from the plasma 5 alpha-dihydrotestosterone-binding protein by electrophoresis on 3.25% acrylamide gels (Rx = 0.5) and on agar gels (anodic migration). It sediments in the 4S region in sucrose gradient containing 0.4 M KCl. Its complex with testosterone dissociates very slowly (t 1/2 = 29 h at 0 degrees C), and is destroyed by heating at 50 degrees C for 30 min and by pronase. Its relative affinities for steroids are 5 alpha-DHT greater than T greater than 5 alpha-androstanediols greater than cyproterone acetate greater than estradiol greater than progesterone. The number of binding sites is limited (about 20 fmoles/mg protein) and the apparent equilibrium dissociation constant (KD) is 5 x 10(-9) M.     

Effect of aminoglutethimide on extraglandular metabolism of exogenous testosterone

Aminoglutethimide (AG), an inhibitor of steroid biosynthesis, seems to have an extraglandular site of action on steroid catabolism. To study this effect, five males with peripheral hypogonadism were first given testosterone propionate and then the same dose was repeated combined with AG and urinary testosterone, and its metabolites were measured. AG was shown to have a very evident effect on the peripheral degradation of exogenous testosterone. This may be responsible for a few signs of virilization and fetal masculinization in women taking AG.     

Competitive binding studies with glucocorticoid receptors from rat-thymus cells: differential temperature-dependence of steroid binding.

Competitive steroid-binding studies were performed with intact rat thymus cells and with cytosol preparations at different temperatures using [1,2-3H]dexamethasone as the labelled ligand. Steroids lacking a 17 alpha-hydroxyl group, such as corticosterone, were better able to compete with [1,2-3H]dexamethasone for binding to glucocorticoid receptors at 0 degrees C than compounds containing a 17 alpha-hydroxyl substituent, such as cortisol. At 37 degrees C the reverse was true. This temperature-dependent change in relative affinities appeared to be unrelated to steroid metabolism or receptor activation, and to depend only on the thermodynamic parameters of the steroid--receptor interaction. Relative biological activities for different steroids agree more closely with the relative affinities determined at 37 degrees C than with those determined at lower temperatures.     

Characterization of the androgen receptor in the anterior pituitary of the rat.

The specific androgen receptors for testosterone (T) and 5alpha-dihydrotestosterone (DHT) in the cytosol fraction of the anterior pituitary of rats have been further characterized using electrophoresis and isoelectric focusing in polyacrylamide gels. After labeling of the cytosol fraction in vivo and in vitro, we were able to demonstrate androgen-protein complexes moving with an electrophoretic mobility (Rf) of 0.5 in 3.25% acrylamide gels containing 0.5% agarose and 10% glycerol. This method was used for quantitative measurements of pituitary androgen receptors, allowing multiple samples to be run simultaneously with little or no non-specific binding. There was no measurable dissociation of the androgen-receptor complexes during electrophoresis. When radioactive testosterone (1 nM) was added to pituitary cytosol fractions in vitro, there was an increase in the binding up to 4 hours of incubation at 0 C and little or no increase between 4 and 24 hours. All the binding studies therefore were done by incubation overnight at 0 C. When cytosol fractions were incubated with increasing concentrations of radioactive testosterone, a typical saturation curve was found. Scatchard plot analysis showed a binding capacity of 12.0 femtomoles/mg protein and the equilibrium constant of dissociation was estimated to be 3.4 +/- 0.7 (SD) X 10(-10)M. Like other androgen-receptor complexes, the testosterone-receptor complex in the anterior pituitary gland had an extremely slow rate of dissociation at 0 C (t1/2 greater than 4 days). The steroid specificity of the cytoplasmic androgen receptors was tested in vitro by the competing efficiency of different unlabeled steroids for [3H] testosterone binding. T and DHT caused the same inhibition of [3H]T to the receptors. However, since metabolism, of DHT to 5alpha-androstane-3alpha,17beta-diol occurred even at 0 C, the affinity of DHT for the receptor is probably somewhat underestimated. Cyproterone acetate had approximately half the affinity for the receptor compared with T, whereas lower affinities were found for progesterone and 17beta-estradiol. Cortisol did not appear to have any affinity for the receptors. Isoelectric focusing in polyacrylamide gels showed a peak of bound radioactivity with an isoelectric point of 5.8. Thus, the characteristics of the cytoplasmic androgen receptors of the anterior pituitary gland are very similar to those of the androgen receptors described in the ventral prostate, epididymis, and testis.     

The estrogen cytosol receptor of female ovine pituitary.

The biochemical parameters of estrone and estradiol binding to the cytosol fraction of ovine anterior pituitary were investigated. When increasing amounts of [3H]estrone or [3H]estradiol were incubated with the 105,000 g fraction from the pituitary, both hormones bound to a receptor with the same apparent KD (mean +/- S.E., estrone = 1.40 +/- 0.30 X 10(-10) M, estradiol = 1.03 +/- 0.11 X 10(-10)M) and the same concentration of binding sites (estrone = 3.22 +/- 0.58 X 10(-14) moles/mg protein, estradiol = 3.92 +/- 0.19 X 10(-14)). No conversion of [3H]estrone to [3H]estradiol under the experimental conditions used could be demonstrated. The receptor-estrogen complex exhibited identical sedimentation coefficients (7-8 S) with either hormone. The receptor was specific only for estrogens; neither 500-fold excess of testosterone nor progesterone affected binding. Competitive inhibition using increasing amounts of non-radioactive estrone or estradiol with [3H]estrone or [3H]estradiol resulted in parallel displacement of the radioactive hormone. These results strongly suggest that both hormones bind to the same pituitary cytosol receptor.     

Progesterone receptor in the hypothalamic cytosol of female rats.

A progesterone (P)-binding component with a sedimentation coefficient of 8S has been demonstrated in hypothalamic cytosol from ovariectomized, estrogen-primed rats using both [H]P-containing sucrose-glycerol gradient analysis and dextran-coated charcoal adsorption of the preisolated 8S component. The association rate constant (K+1) was determined to be 1.90 +/- 0.38 (SD) X 10(6) M-1 min-1 at 0-2 C. The dissociation rate constant (K-1) was 1.86 x 10(-2) min-1, as calculated from the half-dissociation time [37.0 +/- 7.3 (SD) min]. The apparent dissociation constant (Kd) at 0-2 C was determined to be 6-10 nM by Scatchard plot analysis of data obtained from either direct [3HA]P binding or competition of the [3H]P binding by nonradioactive P and by calculating from K-1/K+1. The 8S binding component was protein in nature, and the concentration of binding sites was 12 fmol/mg cytosol protein. On a per U cytosol protein basis, the relative capacities of the specific 8S binding components were: uterus greater than pituitary greater than hypothalamus greater than hippocampus/amygdala greater than cerebral cortex. Competition studies showed a high specificity for P and 5 alpha-dihydroprogesterone. Corticosterone (C), although competing for the binding, had an affinity 8-fold less than P. Implantation of C in adrenalectomized, ovariectomized, estrogen-implanted rats suppressed the 8S binding of [3H]C without affecting the [3H]P binding. The binding of [3H]P to the cytoplasmic 8S component of hypothalamus was greater than that of combined hippocampus and amygdala, while the reverse was observed for the binding of [3H]dexamethasone. These results demonstrate in rat hypothalamic cytosol a tissue and hormone-specific, high affinity, 8S progesterone-binding protein which has many of the properties expected of a hormone receptor.     

Identification of androgen-binding protein from testis cytosol and sertoli cell culture medium of the cynomolgus monkey, Macaca fascicularis

The biochemical and immunological properties of monkey androgen-binding protein (mABP) from the cynomolgus monkey, Macaca fascicularis, have been examined in testis cytosol and medium of primary Sertoli cell-enriched cultures. mABP in testis tissue was separated from the serum protein testosterone-estradiol-binding globulin (mTeBG) by affinity chromatography on Concanavalin A-Sepharose (Con A). mTeBG from serum extracts was completely retained by the lectin and could be displaced with buffer containing alpha-methyl-D-glucoside. In contrast, mABP from testis extracts either did not interact with the Con A and appeared in the void volume or was partially retained by the column and could be eluted with buffer alone. A third component retained by the Con A may represent mTeBG contamination, a form of mABP which binds to Con A, or both. The specific 5 alpha-dihydrotestosterone (DHT)-binding activity in the void volume of the Con A column was designated mABP and was further studied. [3H]DHT binding to mABP was saturable and of limited capacity (0.163 +/- 19 pmol/mg protein). Scatchard analysis of the data was consistent with a single class of binding sites with an apparent dissociation constant (Kd) at 4 C of 2.6 +/- 0.2 X 10(-9) M. DHT was the most effective competitor of [3H]DHT binding to mABP, followed by 2-methoxyestradiol, testosterone, estradiol, and cyproterone acetate. Concentrated Sertoli cell culture medium subjected to steady state polyacrylamide gel electrophoresis produced a single peak of specifically bound [3H]DHT with a mobility similar to that of other androgen-binding proteins. [35S]Methionine-labeled medium proteins were immunoprecipitated with a rabbit anti-human TeBG antiserum. Two bands, corresponding to mol wt of approximately 46,000 and 48,000, were observed by fluorography, with the lighter component being more intense. After androgen affinity chromatography of radiolabeled medium proteins, these two bands were again observed on sodium dodecyl sulfate-urea-containing polyacrylamide gels. These results demonstrate that 1) mABP may be separated from mTeBG by lectin affinity chromatography, as in humans; 2) hTeBG and mABP are antigenically related; and 3) mABP consists of subunits of different mol wt in unequal ratios.     

Effect of 17 beta-estradiol on thyroliberin responsiveness in GH3/B6 rat prolactin cells

GH3/B6 rat prolactin cells were used to analyse at the cellular level the mechanisms by which 17 beta-estradiol (E2) regulates TRH responsiveness of prolactin cells. Before experiments, cells were grown for up to 7 days in 3 different media: normal medium (N) containing 15% horse serum and 2.5% fetal calf serum, CD medium prepared with charcoal-dextran extracted serum and CDE medium supplemented with 4 x 10(-8) M E2. The binding of 3H-TRH (30 min at 37 degrees C) and the TRH-induced percent increase of prolactin release as a function of TRH doses were compared in the 3 conditions. Preculture in E2 enriched medium increased by 50% the number of TRH high-affinity binding sites without modifying their affinity, increased by up to 3 times the percent of the TRH-induced stimulation of prolactin release and improved by one order of magnitude the ED50 of the TRH effect on prolactin release. The presence of HEPES (10 mM) during TRH challenge masked the effect of E2 on the increase in number of binding sites but respected its potentiating effect on prolactin release.     

[3H] Melatonin Binding in Membrane and Cytosol Fractions From Rat and Calf Brain

The binding characteristics of the tritiated pineal hormone, [3H] melatonin, were studied in brain tissues using in vitro binding techniques. In synaptosomal membranes prepared from rat hippocampus and subjected to preincubation at 37 degrees C and multiple washings, high-affinity binding of [3H] melatonin significantly exceeds that previously reported for membrane or cytosol fractions from mammalian brain. Scatchard analysis of saturation binding data indicates that binding affinities are similar in membrane (Kd = 15 nM) and cytosol (Kd = 11 nM) preparations. However, binding is about sevenfold greater in membranes than in cytosol prepared by centrifugation of homogenates at 104,000g for 60 min. Specific binding is also present in both particulate and soluble fractions from calf brain. Inhibition experiments, in rat hippocampal membranes, indicate that norepinephrine is the most potent inhibitor of about 55% of total binding. Serotonin also exhibited high affinity for about 25% of total binding, suggesting that [3H] melatonin labels both adrenergic and serotonergic sites in this brain region. Further studies are required to characterize the serotonergic and adrenergic sites labelled by [3H] melatonin and to determine whether these sites are functionally important receptors for melatonin.     

Androgen metabolism in rat anterior pituitary cells in culture

The metabolism in vitro of 4 androgens, namely testosterone, androstenedione, 5α-dihydrotestosterone and 3α-androstanediol has been studied in male and female rat anterior pituitary cells in primary culture. When testosterone was used as precursor, androstenedione, 5a-dihydrotestosterone and 3α-androstanediol were the main metabolites whereas androstenedione was mainly converted into testosterone, 5α-androstanedione, 5α-dihydrotestosterone and androsterone. Studies on the metabolism of 5α-dihydrotestosterone and 3α-androstanediol showed that these compounds were easily interconverted and were also significantly metabolized to 5α-androstanedione and androsterone. No aromatized compounds could be detected suggesting that androgen action in the pituitary cell occurs directly via the androgen receptor rather than through prior conversion into estrogens.     

Identification of growth hormone binding protein in rat serum

The present report describes the initial characterization of a specific, high-affinity growth hormone binding protein (GH-BP) in adult male rat serum. GH-BP activity was measured by incubation of rat serum with [125I]hGH and [125I]rGH and separation of bound from free GH by dextran-coated charcoal. [125I]hGH binding to rat serum was dependent on serum concentration and incubation time, equilibrium being reached within 10 min both at 4 and 37 degrees C. Binding was rapidly and completely reversible and specific for somatogenic (but not lactogenic) hormones. Scatchard analysis yielded a linear plot with an affinity (Ka) of 1.51 +/- 0.63 x 10(8) M-1. Preliminary data obtained in various physiological conditions showed that GH-BP activity in adult male rats was 5.95 +/- 0.20%/0.1 ml serum. Significantly higher values were obtained in sera of female (21.66 + 0.79%/0.1 ml serum) and pregnant rats (23.02 +/- 1.15%/0.1 ml serum). A closer analysis of these binding values by Scatchard analysis revealed that the binding capacity in pregnant rats (50.5 +/- 5.8 pmol/0.1 ml serum) was significantly higher than in adult female estrous rats (19.2 +/- 6.5 pmol/0.1 ml serum), both being much higher than in adult male rats (2.5 +/- 0.6 pmol/0.1 ml serum). The GH-BP activity of 10-day-old rats was only approximately 63% of the adult male rat value. The presence of high-affinity GH-specific binding protein in rat serum suggests a probable action in regulation of GH activity. The detailed physiological role of rat serum GH-BP is currently being investigated.     

Adenosine receptors in brain membranes: binding of N6-cyclohexyl[3H]adenosine and 1,3-diethyl-8-[3H]phenylxanthine.

N6-Cyclohexyl[3H]adenosine ([3H]CHA) and 1,3-diethyl-8-[3H]phenylxanthine ([3H]DPX) to bind to adenosine receptors in brain membranes. The agonist [3H]CHA has high affinity in both bovine and guinea pig brain (Kd, 0.7 nM and 6 nM, respectively). [3H]CHA binding kinetics are slow (dissociation t1/2;60 min); binding is much higher at 25 degrees C than at 0 degrees C and is inhibited by guanine nucleotides. Potencies of nucleosides and xanthines in competing for [3H]CHA sites indicate that specific binding is entirely to A1 adenosine receptors. In bovine brain, the antagonist [3H]DPX exhibits high-affinity binding (Kd, 5 nM) to the same A1 receptors that bind [3H]CHA. Binding kinetics are rapid (dissociation t1/2, 1 min), and binding is moderately higher at 0 degrees C than at 25 degrees C. In guinea pig brain, [3H]DPX binding has only moderate affintiy (Kd 50 nM), and about 60% of specific binding is to sites that resemble A2 adenosine receptors.     

Opiate receptors are present in the rat testis. Identification and localization in Sertoli cells

We have characterized opioid binding sites in the Sertoli cells of adult and 18-day-old rat testes. Maximal specific etorphine binding was attained after 30 min at 4 C. The binding was reversible, with association and dissociation rate constants of 0.98 X 10(5) M-1 min-1 and 0.33 min-1, respectively. Scatchard analyses and saturation curves revealed a single class of high-affinity, low-capacity binding sites. No opioid binding was observed in Leydig cell cultures. Exposure to opioids for 3 days caused a significant increase in [3H]etorphine specifically bound to the Sertoli cells that was completely prevented by naloxone, demonstrating opioid up-regulation of its own receptor. Chronic opioid treatment of the cultures significantly inhibited androgen-binding protein production, and this effect was prevented by naloxone. Since the circulating concentrations of endorphins (10(-12) M) are lower than the Kd of testis opiate receptors, it is conceivable that opioids of Leydig cell origin act on the specific high-affinity receptors of the Sertoli cells, and may play a role in modulating their function.     

Corticoid receptors in rat brain: evidence for an aldosterone receptor.

The existence of high and low affinity mineralocorticoid-binding macromolecules (receptors) has been demonstrated in vitro in cytosols derived from the adrenalectomized rat brain by the specific binding of [3H]aldosterone (3H-A). The high-affinity aldosterone sites can be distinguished from those sites which have a higher affinity for either [3H]dexamethasone (3H-DM) or [3H]corticosterone (3H-B) on the basis of selectivity for spirolactone SC-9420 or non-radioactive A, DM, and B. The binding of 3H-A to the receptors was maximal after 2 hours of incubation of 0-4C. No significant binding of 3H-A to the receptors could be demonstrated when incubations of the radioactive ligand were performed at either 20 or 37 C, indicating that the receptor is heat-liabile. Scatchard analysis of the 3H-A binding data over a 200-fold concentration range of 3H-A indicated that there are two binding sites for aldosterone, a high affinity component (a1) with a Kd approximately equal to 1.5 X 10(-9)M and a low-affinity component (a2) with a Kd approximately equal to 6.3 X 10(-8)M. A similar study using 3H-DM as the radioactive ligand demonstrated only one site for the 3H-DM binding with a Kd = 6.2 X 10(-9)M. The presence of specific aldosterone receptors in the brain with high affinity, limited capacity, and selectivity for aldosterone suggests a possible extra-renal mechanism of action of the hormone in or mediated through the CNS.     

Characterization of ovarian membrane receptor for 17,20beta-dihydroxy-4-pregnen-3-one,a maturation-inducing hormone in yellowtail,Seriola quinqueradiata

In our previous studies, we tentatively identified 17,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) as a maturation-inducing hormone (MIH) in yellowtail (Seriola quinqueradiata) through in vivo and in vitro experiments. In this study, we investigated the binding sites for radioactive 17,20 beta-P and characterized the receptor binding to the ovarian plasma membrane in yellowtail undergoing first stage of maturation (FSM). Equilibrium binding sites for 17,20 beta-P have been detected within 1h incubation and the binding dissociated completely within 50 min at 4 degrees C and was pH dependent (optimum pH 7.8). Scatchard analyses of specifically bound 17,20 beta-P showed the evidence of a single class of high affinity binding sites (K(D)=22.9 nM), with limited capacity (B(max)=2.1 pmol/g tissue) to the ovarian membrane of yellowtail undergoing FSM. Competition results revealed that ovarian membrane receptor was highly specific for 17,20 beta-P. There was no other steroid competed strongly with the binding sites of [3H]17,20 beta-P, except 17,20 beta-P itself. On the other hand, 17,20 beta-P did not bind to the membrane prepared from maturationally incompetent (MI) and ovulation (OV) stages of oocytes. As the time proceeded after the stimulation of HCG, binding activity increased significantly (0.389+/-0.036 pmol/g tissue) in the ovarian membrane of maturationally competent (MC) oocytes by 12h postinjection. The binding activity was further significant (0.868+/-0.032 pmol/g tissue) at FSM by 24h postinjection and reached its peak (0.920+/-0.115 pmol/g tissue) temporarily at second stage of maturation (SSM) by 36 h postinjection and then sharply declined to the prestimulation levels during OV stage by 48 h postinjection. In addition to our previous findings, the present results indicate that 17,20 beta-P is the MIH in yellowtail.     

Ontogeny of mesenchymal androgen receptors in the embryonic mouse mammary gland

We demonstrate the presence of a high affinity androgen-binding site in the embryonic mouse mammary gland, and describe its appearance and development from the initial formation of the gland bud (day 12) through the androgen-responsive stage (day 14) until term (day 19). Binding assays were done on entire organ rudiments exposed to the ligand [( 3H] testosterone, [3H]5 alpha-dihydrotestosterone, or [3H]methyltrienolone) in vitro, and receptor levels are expressed per gland. In competition experiments, the binding site shows a ligand specificity of a typical rodent androgen receptor. Scatchard analysis yielded a figure of 90-100 million binding sites for one 14-day gland, or approximately 30,000 per target cell. The apparent dissociation constant Kd for ligand binding by the intact tissue was between 0.55 and 0.75 X 10(-9) M. The first binding sites become detectable at the time of mammary bud formation (day 12); their number increases about 20-fold towards the responsive stage (day 14), and they persist at high levels at least until birth. The loss of androgen responsiveness on day 15 is neither accompanied by a loss of receptors nor by a change of their binding affinity.