Barbara C. Eller

Steroid-binding proteins in rabbit plasma: Separation of testosterone-binding globulin (TeBG) from corticosteroid-binding globulin (CBG), preliminary characterization of TeBG, and changes in TeBG concentration during sexual maturation.

Two distinct steroid-binding proteins are present in rabbit plasma. One of the proteins (TeBG) binds [3-H]5 alpha-dihydrotestosterone (5 alpha DHT) and [3-H]testosterone. The affinity of this binding protein for 5 alpha DHT was 3-4 times greater than for testosterone. Binding of [3-H]5 alphaDHT could be inhibited by unlabeled 5 alpha DHT, testosterone, 5 alpha-androstan-3 alpha,17 beta-diol (3 alpha-diol), and 17 alpha-methyl- B-testosterone (skf) 7690). The relative affinity of the competitors was: 5 alpha DHT greater than 3 alpha-diol greater than testosterone greater than SKF 7690. The antiandrogens, cyproterone (1,2 alpha-methylene-6-chloro-pregn-4,6-diene-17 alpla-ol-3,20 dione), cyproterone-17-acetate, and 6 alpha-bromo-17 beta-hydroxy-17 alpha-methyl-4-oxa-5 alpha-androstan-3-ine (BOMT) were ineffective in competing for [3-H5d alpha DHT binding sites, as were 4-androstene-3, 17-dione, 17 beta-estradiol (E2), progesterone, and cortisol. The formation of the [3-H]5 alpha DHT-TeBG complex was extremely rapid; the binding reaction was essentially completed in 15 s. The complex dissociated rapidly in the presence of charcoal. The dissociation rate constant (Kdiss) was 0.157 min- minus 1 and the dissociation half-time t-1/2) was 4.5 min. In the presence of charcoal and unlabeled 5 alpha DHT the Kdiss was 0.268 min- minus 1 and the t=1/2 was 2.6 min. The sedimentation coefficient of TeBG was congruent to 4.6 S and its molecular weight, estimated by gel filtration on a calibrated Sephadex G-200 column, was congruent to 75,000. The concentration of TeBG in male rabbit plasma decreased with sexual maturation and was approximately three times higher in adult females than in adult males. The other protein (CBG) bound both [3-H]cortisol and [3-H]progesterone. Binding of these compounds could be inhibited by unlabeled cortisol and progesterone, but not by unlabeled 5 alpha DHT, testosterone, or E2. CBG had a sedimentation of congruent to 3.9 S and an apparent molecular weight of congruent to 105,000. TeBG could be separated from CBG by a 60% ammonium sulfate precipitation and by gel filtration chromatography. Both proteins are thermolabile; TeBG is inactivated at temperatures above 30 degrees C and CBG is inactivated at temperatures above 50 degrees C.

Studies on the site of origin of the androgen binding protein present in epididymal cytosol from mature intact rabbits

Abstract An examination of the site of origin of the 17β-hydroxy-5α-androstan-3-one (5αDHT) binding protein that exists in cytosol prepared from the caput epididymidis from sexually-mature intact rabbits was approached in the following way: Rabbits a) had the ductuli efferent es unilaterally ligated, b) were hemicastrated, c) were made unilaterally cryptorchid. These procedures were designed to decrease or eliminate the flow of testicular fluid into the epididymis. When this occurred, there was a marked decrease or total elimination of 5αDHT binding to cytosol prepared from the caput from the experimental epididymis, as compared to cytosol from the contralateral control side. These results suggest that the 5αDHT binding moiety in cytosol prepared from the caput epididymidis from intact sexually-mature rabbits is of testicular origin and is not the target tissue “receptor.”

Estradiol binding in cytosol from epididymides of immature rabbits

A highly specific, high affinity binding protein for estradiol-17beta (E2) is present in cytosol prepared from the epididymides of immature (21-53 day old) rabbits. This binding moiety sediments on sucrose gradients as an 8S species under low ionic strength conditions and as a 4S species under conditions of high ionic strength (0.3 M KCL). The relative binding affinities of estrogens for the binding protein was E2 is greater than estrone is greater than estriol. Neither 5alpha-dihydrostestosterone (5alphaKHT), progesterone, nor cortisol were able to inhibit binding of [3H]E2 to epididymal binding sites. An 8S binding moiety for E2 was present in testicular cytosol but not in muscle. An apparently non-specific binding component for E2 was present in plasma which sedimented in the 4S region of low ionic strength gradients. The epididymal E2 binding moiety was distinct from a 4S androen binding protein of testicular origin which is detectable in cytosol prepared from epididymides of rabbits at certain stages development. We were unalbe to detect a specific E2 binding protein in epididymal cytosol from mature intact or 4-day castrated rabbits. The E2 binding component in the cytosol of immature rabbits had an Kd congruent to 2-10 X 10-10 M and the concentration of binding sites was in the order of 1-4 X 10-13 mmoles/mg of protein. The binding component was thermo-labele and pronase, but not nuclease, sensitive.

Identification of cytoplasmic estrogen receptors in the accessory sex organs of the rabbit and their comparison to the cytoplasmic estrogen receptor in the epididymis

Estrogen receptors are present in cytosol prepared from the accessory sex organs (vesicular gland, proprostate, prostate, bulbourethral gland) of sexually immature and of sexually mature rabbits. The receptor in these organs from animals of both age groups has a sedimentation coefficient of 8-10S on low ionic strength (0.01 M KCl) sucrose gradients. Under high ionic strength (0.4 M KCl) conditions, the receptor sediments at approximately 4S. The cytoplasmic estrogen receptor from the epididymis shows age-dependent changes in its sedimentation coefficient. It is 8S under low ionic strength conditions when prepared from immature rabbits and 4S under identical conditions when prepared from sexually mature animals. Although the dissociation constant of the cytoplasmic estrogen receptor in the immature and mature epididymis and accessory sex organs remains constant during development (approximately 0.1 nM), the number of available cytoplasmic estrogen binding sites declines from about 160 fmoles/mg cytosol protein in the immature rabbit to about 40 fmoles/mg cytosol protein in the adult animal. The estrogen receptor in the accessory sex organs is highly specific, the relative affinities of various potential competitors being: estradiol and estrone = 1, diethylstilbestrol = 0.3, estriol = 0.2, tamoxifen = 0.08, testosterone = 0.0004 and 5 alpha-DHT = 0.00005. Changes with age in the physicochemical characteristics of the estrogen receptor and in the concentration of binding sites suggest that the estrogen receptor may be involved in the development and physiological regulation of the male reproductive tract.

Nuclear binding of [3H]-androgens by the epididymis of sexually mature castrated rabbits.

Abstract We have demonstrated that when minces of epididymides from castrated rabbits are incubated with [3H]-testosterone radioactivity bound to a macromolecular component, it enters the nuclei. This entry is a temperature dependent phenomenon which is greater upon incubation at 23°C than at 0°C. In contrast, no macromolecular bound hormone could be detected in leg muscle cytosol or nuclei. The amount of radioactivity extractable from epididymal nuclei was a function of the molar concentration of KC1 in the extraction medium. However, even when 1.0 M KC1 was used, greater than 30% of the radioactivity bound to nuclei remained unextractable. Of the naturally occurring steroids that we used (testosterone, estradiol-17β, and cortisol) in competition studies, unlabeled testosterone was the most effective in preventing the nuclear accumulation of labeled androgen. Estradiol-17β also was effective in this regard, but was much less potent than testosterone under the conditions employed. Unlabeled cortisol was an ineffective inhibitor. The antiandrogens, cyproterone and SKF 7690, both of which inhibit binding to the epididymal cytoplasmic androgen receptor, completely inhibited nuclear accumulation of radioactivity, implying that nuclear accumulation is dependent on an initial binding to the cytoplasmic receptor. We have also demonstrated that the rabbit epididymis is capable of converting testosterone to 5α-dihydrotestosterone (5α-DHT). Although the tissues were incubated with [3H]-testosterone, 5α-DHT was selectively bound by the cytoplasmic receptor and concentrated in the nuclei.

Androgen binding to cytosol prepared from epididymides of sexually mature castrated rabbits: Evidence for a cytoplasmic receptor ☆

The presence of androgen-binding activity in cytosol prepared from the major anatomical segments (caput, corpus, and cauda) of the epididymis of castrated sexually mature rabbits has been demonstrated. A portion of this binding activity is likely to be the epididymal androgen receptor. When epididymal cytosol from adult castrated rabbits is analyzed on low-ionic strength (0.01 MKCl) sucrose gradients, two peaks of macromolecular binding could be detected, one congruent to 4.6S and one congruent to 8S. On gradients containing 1.0 M KCl, only one sedimenting form congruent to 4.6S could be demonstrated, suggesting that the 8S component is composed of aggregates. If cytosol was preincubated with labeled androgen, followed by an incubation with unlabeled androgen, and subsequently analyzed for binding on low-ionic strength gradients, only the congruent to 8S peak could be detected, indicating that most of the binding in the congruent to 4.6S region was rapidly dissociable. This suggests that binding in this region was to moieties other than receptor. Since androgen binding proteins (ABP) of testicular origin would have been cleared from the epididymis at the timepoints that we concentrated on for most of these studies, the 4.6S binding probably represents the association of androgen with plasma testosterone binding globulin (TeBG). The binding of androgen to the receptor can be inhibited by cyproterone, while this antiandrogen does not inhibit binding to either ABP or TeBG at the concentration used.

Changes in 5α- dihydrotestosterone binding to epididymal cytosol during sexual maturation in rabbits: Correlation with morphological changes in the testis and epididymis ☆

Abstract We previously demonstrated that the caput epididymis of intact sexually mature rabbits contains a specific high-affinity binding protein for 5α-dihydrotestosterone (5αDHT). The other anatomical segments (corpus and cauda) of the epididymes of these animals had no detectable 5αDHT-binding activity. We have further shown that this binding was due to an androgen-binding protein of testicular origin. In the present study we have investigated 5αDHT binding to epididymal cytosol from sexually immature rabbits (20−104 days old). Using sucrose gradient ultracentrifugation, we have detected a unique pattern of binding. The pattern correlated well with testicular and epididymal maturation, but there was little correlation with chronological age or body weight. In the most immature animals (Group I) the seminiferous tubules appsared as solid cords and the epithelium of the ductus epididymis consisted of a single row of columnar cells. Epididymes from this group had little or no detectable 5αDHT-binding activity. In the second group (Group II), there was 5αDHTbinding to all three segments. The seminiferous tubules of these rabbits exhibited spermatogenic activity and lumen formation. The height of the epididymal epithelium had increased uniformly throughout the duct. The third group (Group III) had 5αDHT-binding only in caput cytosol. Spermatogenesis had progressed to the formation of elongated spermatids in the most immature animals of this group to the release of spermatozoa in the most mature ones. The caput epithelium of this last group of rabbits was fully differentiated. Unilateral orchidectomy of Group II rabbits resulted in a decrease in [3H]5αDHT-binding activity on the operated side as compared to the contralateral non-operated control side, suggesting the testicular origin of the binding protein. The failure of cyproterone or cyproterone acetate to inhibit [3H5αDHT-binding to the protein, the lack of effect of N-ethylmaleimide on binding, and the rapid dissociation rate of the[3H5αDHT-binding protein complex suggested that the binding moiety was testicular androgen-binding protein (ABP).

High affinity estrogen binding by rabbit liver

Macromolecular binding components for [3H]estradiol-17beta are present to cytosol prepared from rabbit liver. When cytosol from sexually mature male liver was incubated with [3H]estradiol and analyzed for binding on low ionic strength sucrose gradients, two peaks of binding activity were detected. One peak had a sedimentation coefficient of 4--5 S and the other had a sedimentation coefficient of 8--9 S. The two components differed from each other regarding steroid specificity and various physiocochemical parameters. [3H]estradiol binding to the 4--5 S component was not inhibited by estrogens, 5alpha-dihydrotestosterone, progesterone or cortisol. Binding to this component did not appear to be saturable and label was rapidly stripped from it by charcoal. Estradiol binding to the 8--9 S component was estrogen specific, saturable and of high affinity. The specific binder dissociates on high ionic strength sucrose gradients and sediments as a 4--5 S moiety. The specific binding protein has a Kd of 3.05 . 10(-10) M and a dissociation half-time of 33 h and there are 35.2 fmol of binding sites/mg cytosol protein. Estrogen binders are also present in liver cytosol from sexually mature female and sexually immature male rabbits. During prolonged incubation of [3H]estradiol with mature male liver cytosol at 0--5 degrees C polar metabolites of estradiol are produced.

Hormonal effects on the estrogen receptor system in the epididymis and accessory sex organs of sexually immature rabbits

The epididymis and male accessory sex organs (vesicular gland, prostate, and bulbourethral gland) of sexually immature rabbits contain a functional estrogen receptor system which is regulated in an organ-specific manner by various hormones. In both intact and castrated animals, acute estrogen challenge causes depletion of estrogen receptor from the cytosolic fraction and its appearance in the nuclear fraction of these tissues. A considerable amount of unoccupied nuclear receptor was detected both before and after estrogen challenge. An estrogen-activated, receptor-processing mechanism is operable in these organs since chronic treatment (daily for 14 days) with estradiol benzoate modified the levels of total estrogen receptor, and altered the relative amounts of occupied to unoccupied nuclear receptor present following estrogen challenge. Chronic treatment with estradiol benzoate, Tamoxifen, and testosterone propionate (alone and in combination) had differential, organ-specific effects on the ability of subsequent estrogen challenge to cause accumulation of nuclear receptor. The vesicular gland was the most responsive to estrogen treatment and the bulbourethral gland the least responsive.

Androgen metabolism by mature rabbit epididymal tissue: The effects of castration and androgen replacement

Abstract Castration of adult rabbits leads to a rapid decline in the ability of the epididymis to metabolize androgens. Castrated rabbits were injected with testosterone propionate (T.P.) (2 mg/kg/day) to determine if androgen administration would result in the maintenance of epididymal 4-ene-3-ketosteroid 5α-reductase and 5α-androstane-3α(3β)-hydroxysteroid dehydrogenase. These enzymes are required for the conversion of testosterone to 5α-dihydrotestosterone (5α-DHT) and 5α-DHT to 5α-androstane-3α-17β-diol (3α-diol), respectively. This dose of T.P. resulted in plasma levels of testosterone that were 5–7 fold higher than and in epididymal levels of testosterone that were similar to non-treated intact rabbits. When epididymal minces from T.P.-treated castrated rabbits were incubated with 8.25 × 10 −9 M [ 3 H]-testosterone for 2 h at 0°C, followed by l h at 23°C, analysis of metabolites formed indicated that the post-castration induced decline in 5α-DHT production did not occur and the 3α-diol synthesis was slightly elevated. However, when epididymal minces were incubated with [ 3 H]-testosterone at 32°C for 2h, there was a 3-fold increase in both 5α-DHT and 3α-diol formation as compared to the above incubation conditions. Castration resulted in an 80% decrease in the amount of 5α-DHT produced. Although androgen replacement failed to maintain 5α-DHT synthesis at control levels, the amount of 5α-DHT synthesized was twice that of castrated non-treated rabbits. There was no apparent effect on the amount of [ 3 H]-testosterone that was converted to 3α-diol. When epididymal minces were incubated with [ 3 H]-5α-DHT for 2 h at 32°C, 3α-diol formation was significantly greater ( P 3 H]-testosterone. Castration leads to a 50% decline in 3α-diol synthesis. Androgen replacement resulted in a significant stimulation ( P