Androgen binding protein (ABP) in rabbit testis and epididymis

ABP has been measured in 105,000 g supernatants of testis and epididymls from rabbits of different ages and compared with a similar androgen binding protein (TeBG) in rabbit serum. Whereas the concentration of ABP in the caput epididymidis increased markedly from immaturity to adulthood, serum TeBG decreased, indicating that ABP and TeBG are regulated by different hormonal mechanisms.The concentration of ABP (pmoles/mg protein) in sexually mature rabbits was much higher in the epididymis than in the testis. Within the epididymis most of the ABP was concentrated within the caput, and very low amounts were found in the cauda, indicating that binding activity of ABP is destroyed as it passes through the epididymis.In addition to ABP (R_f ~0.7), rabbit epididymal supernatant contains a larger binding protein for 5α-dihydrotestosterone (DHT; 17β-hydroxy-5α-androstan-3-one) with slower electrophoretic mobility (R_f ~0.4) and a more rapid sedimentation rate on sucrose gradients (7S). This component is most probably the intracellular androgen receptor in the rabbit e pididymis.     

Intracellular receptors for 5alpha-dihydrostestosterone in the epididymis of adult rats. Comparison with the androgenic receptors in the ventral prostate and the androgen binding protein (ABP) in the testicular and epididymal fluid

Epididymal cytosol fractions of adult short-time castrated rats contained at least two different androgen protein complexes by experiments $\text{in vivo}$ (Complex I and II).Complex I is probably located intracellularly in the epididymal cells. It was specific for 5α-dihydrotestosterone (DHT) and appeared to be very similar to the cytoplasmic DHT-receptor complexes in rat ventral prostate. By ultracentrifugation on sucrose gradients, it sedimented as heavy aggregates 8–10 S complexes and 3–4 S complexes, which dissociated into 3–4 S complexes at high ionic strength. Complex I was eluted in the void volume from columns of Sephadex G-200.Complex II was also specific for DHT and showed physical properties similar to those of the androgen binding protein (ABP) in the testicular fluid. It was eluted between immunoglobulin G (IgG) (53 Å) and albumin (36 Å) by gel filtration on Sephadex G-200. The sedimentation coefficient was 4.5–5 S (mean 4.6 S_W, 20) at both high and low ionic strength.Complex I and the cytosol receptors for DHT in the rat ventral prostate were both destroyed by heating at 50° C for 30 min, addition of 1 mM p-chloro-mercuri-phenyl-sulphonate (PCMPS) and charcoal absorption (1 mg/mg protein) overnight, whereas complex II was not influenced by similar treatment.Hemi-castration for 4 weeks caused complex II to disappear completely from the castrated side, confirming the intraluminal localization of this complex. Complex I was not influenced by such treatment, indicating that this protein is located within the epididymal cells. The similarity between complex I and the cytoplasmic DHT-receptor complexes in the ventral prostate also suggests that complex I represents the cytoplasmic receptors for DHT in the epididymis.     

Estradiol-17 beta inhibition of androgen uptake, metabolism and binding in epididymis of adult male rats in vivo: a comparison with cyproterone acetate

The effects of estradiol-17 beta on androgen uptake, metabolism and binding were studied in rat epididymis in vivo in comparison with cyproterone acetate. Steroids (250 ug/100 g body weight) were injected 5 min prior to 3H-testosterone in castrate rats. Estradiol-17 beta inhibited 3H-testosterone uptake into epididymal cytosol by 58% as compared to 38% by cyproterone acetate. 3H-Testosterone uptake into epididymal nuclei was inhibited 95% by estradiol-17 beta and 83% by cyproterone acetate. Total bound radioactivity in cytosol fractions was reduced to a greater extent by estradiol-17 beta than cyproterone acetate when either 3H-testosterone or 3H-dihydrotestosterone was injected. Binding of 3H-dihydrotestosterone to nuclear receptors was completely abolished by estradiol-17 beta; whereas approximately 20% binding remained in the nuclear extract after cyproterone acetate treatment. Metabolism of 3H-testosterone in vivo was also altered by estradiol-17 beta, resulting in diminished conversion to 3H-dihydrotestosterone. Cyproterone acetate, on the other hand, did not affect 3H-testosterone metabolism. Estradiol-17 beta and cyproterone acetate inhibited in vitro binding of 3H-dihydrotestosterone to the intracellular cytoplasmic receptor, but not the intraluminal androgen binding protein (ABP). These data suggest that estradiol-17 beta may have a more potent antiandrogenic effect on the epididymis than cyproterone acetate due to inhibition of 5 alpha reduction of testosterone as well as binding to the androgen receptor.     

A protease acting on the estrogen receptor may modify its action in the adult rabbit epididymis

We have previously shown that the cytosolic estrogen receptor in adult rabbit epididymides sediments as an congruent to 3 S species on sucrose gradients containing 0.01 M KCl while that from immature rabbit epididymides sediments at congruent to 9 S. This age-dependent decrease in sedimentation coefficient is attributable to the appearance of a leupeptin-sensitive protease as the animals mature. We now show that if adult epididymides are homogenized in buffer containing leupeptin, the 9 S receptor can be demonstrated, indicating inhibition of receptor degradation. In vitro nuclear uptake studies conducted in the absence of leupeptin indicated that the proteolyzed receptor was not an efficient nuclear binder. When leupeptin was present, nuclear uptake increased 6-fold and it was accompanied by depletion of receptor from the cytosol. Binding of the receptor to nuclei was specific since it could be inhibited by unlabeled estrogens but not by unlabeled 5 alpha-dihydrotestosterone or progesterone. In vitro mixing experiments indicated that the proteolytic activity was associated with the crude nuclear fraction since, in the absence of leupeptin, they had reduced ability to bind estrogen receptor present in immature epididymal cytosol. Specific in vivo binding of [3H]estradiol by adult and immature rabbit epididymides could be demonstrated. The time course of in vivo binding of [3H]estradiol by adult rabbit epididymal nuclei indicated maximum binding (70 fmol/g tissue) at 30 min following injection. By 60 min, the amount of binding had decreased to about 25 fmol. The accessory sex organs, which do not contain the protease, also exhibited maximum binding (150 fmol/g tissue) at 30 min. However, at the 60 min period binding was still about 140 fmol. Processing the tissues in buffers containing leupeptin had no effect on the results obtained. These results are interpreted to indicate that the presence of the protease decreases nuclear binding of the estrogen receptor and shortens nuclear occupancy. This combination of factors may be responsible for the decrease in estrogen action in the adult rabbit epididymis.     

Interaction of medroxyprogesterone acetate with cytosol androgen receptors in the rat hypothalamus and pituitary

The binding of medroxyprogesterone acetate (MPA) with cytosol androgen receptors from rat pituitary and hypothalamus was studied. The pituitary and hypothalamic cytosol androgen receptors from adult castrated female rats were in vitro labeled using 3H natural (testosterone (T) and 5 alpha-dihydrotestosterone (DHT] and [3H]synthetic (methyltrienolone) androgens as radioligands. The [3H]androgen-receptor complexes sedimented with a coefficient of 8S in linear sucrose gradients. When incubated with an excess of radioinert MPA, specific binding was abolished indicating interaction of MPA with androgen receptors. Furthermore specific [3H]MPA-androgen cytosol receptor complexes could be identified in these neuroendocrine tissues when a post-gradient receptor labeling technique was used in the absence or presence of radioinert MPA, DHT, and triamcinolone acetonide. A study of binding kinetics disclosed that the equilibrium dissociation constant and saturation binding capacity for the MPA binder, were similar to those exhibited by DHT binding to androgen receptors in both studied tissues under identical experimental conditions. The overall results were interpreted as demonstrating that MPA interacts with cytosol steroid receptors other than those of progesterone in the rat hypothalamus and anterior pituitary. The data are consistent with MPA binding to androgen receptors.     

An androgen binding protein in the testis cytosol fraction of adult rats. Comparison with the androgen binding protein in the epididymis

The cytosol fraction of rat testicular homogenates contained a specific androgen binding protein (t-ABP), indistinguishable from, the androgen binding protean in. the epididymis(e-ABP) in terms of its chromatographic behaviour by gel filtration, sedimentation rate, mobility on polyacrylamide electrophoresis and steroid specificity(5α-diaydrotestosterone(DHT) > testosterone > estradiol-17β > parogesterone and estradiol-17α).The stability of t-ABP as well was similar to that of e-ABP. The aBP-DHT complexes were stable between pH 6.5–10, exhibiting the highest degree of binding between pH 8–9. The binding activity persisted after heating at 50°C for 30 min., whereas heating at 60°C for 30 min. completely destroyed the binding. DHT did not significantly increase the stability towards pH and temperature denaturation. Binding activity was not reduced by 1 mM p-chloro-mercuri-phenyl-sulphonate (PCMPS), whereas similar treatment with 1 nM N-ethyl-maleimide(NEM) or 1 mM Ellman's reagent reduced it by some 10–50 per cent. Exposure to 10 mM β-mercaptoethanol(βME) reduced the binding by 60–70 per cent. These studies strongly indicate that t-ABP and e-ABP are identical proteins.Hemicastration for 4 weeks eliminated the ABP from the epididymal cytosol fraction on the castrated side, indicating that this protein is produced by the testis.     

Tissue androgens and androphilic proteins in rat epididymis during sexual development

The appearance of the epididymal 8s cytoplasmic receptor in the rat during sexual development was followed and correlated with the endogenous concentrations of three biologically active androgens in the epididymis, testosterone (T), dihydrotestosterone (DHT)and 5α-androstane-3α, 17β-diol (Diol).The results indicate that receptors could not be evidenced in the 10-day-old animal in which androgens were undetectable. The 8s receptor was first detected in the 20 day-old rat, coincidently with a rise in the concentration of DHT (0,24 ng/ epididymis) while T and Diol remained too low to be measured. At the 26th day of life a peak of androgen binding activity, sedimenting at approximately 4S, was seen and probably corresponds to ABP. This pattern of two binding peaks (8 and 4S) remained constant through adulthood. The association constant (Ka) of the 8s receptor from 35-, 45- and 60-day-old rats was found to be similar.T became detectable in the epididymides of the 35 day-old rat (0.03 ng/ep.) and increased with sexual maturation. However, DHT concentrations were higher than those of T at all ages studied and Diol could not be detected at any age.These results raise the possibility that the synthesis or availability of receptors in the developing animals is under androgenic control.     

Developmental changes in and hormonal regulation of estrogen and androgen receptors present in the rabbit epididymis

Both androgen and estrogen receptors (AR and ER) are present in the rabbit epididymis. We have used the sucrose gradient method to examine receptor sedimentation properties, receptor concentration, and distribution of receptors among the caput, corpus, and cauda of the epididymis to determine changes that occur in these parameters as the animals age. The 9S form of the ER is present in all three epididymal segments of the immature rabbit, with the highest concentration occurring in the cauda. The 8.2S form of the AR is also present in all three segments of the immature epididymis, with the highest concentration occurring in the caput. Short-term castration (3 days) leads to an increase in the amount of both AR and ER detected. ER are present in all segments of the immature epididymis at higher concentrations than AR. The functional 9S form of the ER disappears as the animals mature, the result of a tissue-specific protease that our laboratory previously has shown proteolyzes ER to a non-DNA-binding 3.8S form. Long-term castration (3 mo) of adult rabbits results in the reappearance of the 9S form of the ER in all segments of the epididymis. The reappearance of the 9S form of the ER is also seen in animals castrated for 1 mo, but not in those castrated for 2 wk. Administration of testosterone once daily for 2 wk to adult animals castrated for 6 wk results in the disappearance of the 9S form of the ER and the reappearance of the 3.8S form, suggesting that the tissue-specific protease is androgen-dependent. In this way, circulating androgens may play a role in regulating the concentration and form of the ER in the rabbit epididymis. There is little change in the concentration or distribution of AR in the epididymis of adult rabbits castrated for 3 mo as compared to those castrated for 3 days. This implies that circulating androgens are not required for maintenance of AR in the epididymis. Our data demonstrate that there are temporal differences in the presence and concentration of ER and AR in the epididymis and suggest that there is a differential, age-dependent regulation of the development and function of the epididymis by androgens and estrogens.     

Castration induces changes in the cation‐dependent mannose‐6‐phosphate receptor in rat epididymis: Possible implications in secretion of lysosomal enzymes

It is believed that the mammalian epididymis participates in the maturation of the sperm due to its secretory activity. High concentrations of several secreted acid hydrolases are found in the epididymal lumen. Moreover, some of these enzymes are secreted by the epididymal epithelium in an androgen‐dependent fashion. In this study, we attempted to discern whether mannose‐6‐phosphate receptors (MPRs) regulate transport and secretion of lysosomal enzymes in the rat epididymis, and if these events are altered when the animals are subjected to hormonal manipulation. We observed that expression of cation‐dependent MPR (CD‐MPR) and cation‐independent MPR (CI‐MPR) increased significantly in caudal epididymis of castrated rats by immunoblot. This increase was corroborated by quantitation of MPRs, by binding assays. This change could be due to androgen deprivation, as a similar effect was observed after treatment with the anti‐androgenic drug flutamide. Furthermore, we observed that the CD‐MPR was redistributed to the apical area of the epithelium on castrated rats by immunohistochemistry, which is compatible with the redistribution of the receptors toward lighter fractions in a Percoll gradient. Consistent with a possible involvement of the CD‐MPR in the secretion, we observed an increase in pro‐cathepsin D levels in epididymal fluid after castration. We conclude that the CD‐MPR might be regulated by hormones and that this receptor might be involved in the secretion of specific enzymes into the rat epididymis. J. Cell. Biochem. 110: 1101–1110, 2010. Published 2010 Wiley‐Liss, Inc.     

The effects of various steroids on testosterone metabolism by the sexually mature rabbit epididymis

We examined the influences of steroids present in the epididymis on androgen metabolism by epididymal tissue and on the binding of androgen metabolites to the epididymal androgen receptor in castrated adult rabbit epididymides under in vitro conditions. The conversion of [3H]testosterone to [3H]17 beta-hydroxy-5 alpha-androstan-3-one (5 alpha-DHT) and to [3H]5 alpha-androstane-3 alpha (beta), 17 beta-diol was inhibited by unlabeled steroids in the following manner progesterone greater than testosterone greater than estradiol. Unlabeled 5 alpha-DHT did not inhibit [3H]testosterone metabolism indicating that product inhibition is not an important regulatory event. The antiandrogen cyproterone acetate did not inhibit the formation of 5 alpha-reduced metabolites of [3H]testosterone. All of the compounds used inhibited androgen binding to the classically defined cytoplasmic and nuclear androgen receptor.     

Identification of an androgen receptor in the adult chicken oviduct

The chicken oviduct androgen receptor was characterized by sucrose density gradient centrifugation, Scatchard analysis, competition studies, and affinity labeled with dihydrotestosterone 17 beta-bromoacetate. A specific 8.5 S peak was seen on 0.01 M KCl sucrose density gradients when the receptor was labeled with [3H]5 alpha-dihydrotestosterone. Specific 4.6 S peaks were seen when receptor labeled with [3H]5 alpha-dihydrotestosterone or [3H]dihydrotestosterone 17 beta-bromoacetate was analyzed on 0.3 M KCl sucrose density gradients. Scatchard analysis of [3H]5 alpha-dihydrotestosterone binding by oviduct cytosol was consistent with two binding sites. A Kd of 0.13 nM was found for the high affinity androgen receptor. Competition studies showed the following order of ligand affinity: 5 alpha-dihydrotestosterone greater than dihydrotestosterone 17 beta-bromoacetate greater than progesterone greater than estradiol. A 61.2 kDa protein was specifically covalently labeled with [3H]dihydrotestosterone 17 beta-bromoacetate. The chicken oviduct androgen receptor possesses characteristics similar to other androgen receptors, and provides a good source of androgen receptor for physicochemical studies of the native receptor protein.     

Characteristics of androgen binding in rat adrenal tissue using [3H]methyltrienolone (R1881) as tracer

A high level of binding of [3H]methyltrienolone (R1881 = 17beta-hydroxy-17alpha-methyl-estra-4, 9, 11-trien-3-one) was found in cytosol prepared from adrenals of castrated male rats. Binding of [3H]R1881 was of high affinity (DK = 6.2 nM) and highly specific for androgens. The [3H]R1881 complex migrates at 7-9S on sucrose gradients in low ionic strength buffer and at 4-5S in buffer containing 0.4M KC1. All binding studies have been performed in parallel with rat ventral prostate and adrenal cytosol. The present data suggest the presence of an androgen binding component in rat adrenal tissue.     

Some effects of breeding season and castration on the prostate and epididymis of the brushtail possum, Trichosurus vulpecula

In an attempt to understand the mechanism(s) responsible for the reported marked seasonal increase in prostatic, but not epididymal, weight in T. vulpecula, a number of parameters were measured in tissues from mature, entire males sampled within and outside of the breeding season and from castrates. Conditions for the measurement of cytosol androgen receptors were also established. The weight of both the prostate and the epididymis was significantly elevated in the breeding season but the relative increase in prostate weight was considerably greater. The increase in prostatic weight was associated with a decrease in DNA: g tissue and an increase in protein: DNA and RNA: DNA ratios, each indicative of cellular hypertrophy and/or accumulation of secretory product. In the epididymis there were no significant seasonal changes in RNA: DNA, protein: DNA or DNA: g tissue ratios. Low-capacity, high-affinity binding was demonstrated in the epididymal and prostatic cytosols and values for the equilibrium association constants and receptor concentrations were within the range reported for androgen receptors in eutherian species. The temperature sensitivity of the binding, steroid specificity and slow dissociation in the cold indicated that in both tissues cystosol receptor and not androgen-binding or serum-binding protein(s) were being measured. In prostatic, but not epididymal, cytosol a low level of progesterone binding was observed and was masked by triamcinolone acetonide. When expressed in terms of tissue DNA, cytosol androgen receptor level in the prostate only was elevated in the breeding season. Prostatic tissue showed a low level of 5 alpha-reductase in vitro which was not influenced by season. However, both tissues showed a high concentration of 5 alpha-dihydrotestosterone and in the prostate, where seasonal effects were measured, the concentration was higher in the breeding season. This indicates that although 5 alpha-dihydrotestosterone is the likely active androgen in the prostate it may be formed elsewhere. Part of the explanation for the increased growth of the prostate in the breeding season appears to be a change in receptor concentration coupled with elevated tissue androgen level.     

Medroxyprogesterone acetate has opposite effects on the androgen binding protein concentrations in serum and epididymis

In the rat, the effects of progestin and androgen administration on serum, testicular and epididymal androgen binding protein (rABP) concentrations were determined and related to the organ weight and morphology. Adult rats were treated with medroxyprogesterone acetate (MPA; 17 alpha-acetoxy-6 alpha-methylprogesterone), testosterone propionate (TP) and mibolerone (MB; 7 alpha, 17 alpha-dimethyl-19-nortestosterone). MPA reduced testicular and epididymal weights and the concentrations of serum follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone. During MPA treatment testicular and epididymal ABP content declined in parallel with organ weights and hormone concentrations, whereas serum ABP concentrations increased. Combinations of MPA and TP reduced testicular and epididymal ABP, but the reductions were less than with MPA alone; this combined treatment also elevated serum AMP. Both MB and TP reduced ABP in the male reproductive tract, but unlike MPA did not increase the concentration of this protein in serum. The results suggest that MPA acts directly on Sertoli cells resulting in increased ABP release into the blood. The comparison was made of steady state polyacrylamide gel electrophoresis (SS-PAGE) and radioimmunoassay (RIA) methods of estimating rABP. The potency ratio of testicular ABP estimated by the two methods (RIA:SS-PAGE) was three times higher than this ratio in the epididymis in normal and hormonally treated animals. Due to differences in end points, these observations imply that these assays do not quantify the molecules in the same way in one or both of these tissues. The results indicate, however, that both assays are suitable for following rABP concentration in animals with altered hormonal states.     

Reversible interaction between androgen binding protein and testicular macromolecules causing inhibition of androgen binding activity.

Sertoli cells in culture isolated from immature rat testes secrete androgen binding protein (ABP) in the culture medium. Binding activity of ABP in concentrated medium was estimated with equilibrium dialysis against 1 nM dihydrotestosterone at 4 degrees C. The ABP protein activity was inhibited approximately 50% through addition of cytosol preparations from testis or liver, but not from brain tissue, to the concentrated culture medium; this inhibition remained constant for at least two days. The inhibitor is probably a macromolecule, because the activity could not be removed by charcoal treatment and dialysis. The percent inhibition of ABP binding activity was increased when increasing amounts of cytosol were added, it decreased in the presence of increased concentrations of androgens, but it was not influenced by variations of the concentration of ABP. Inhibition of androgen binding to ABP by cytosols in the presence of 1 nM testosterone could be reversed after dialysis in the presence of 10 nM testosterone. These results suggest a reversible competition between testosterone and the testicular macromolecule for ABP. The occurrence of this interaction between ABP and a testicular macromolecule can explain the variable results of estimated ABP binding activity in testis cytosol preparations.     

Interconversion of androgen receptor forms by divalent cations and 8 S androgen receptor-promoting factor. Effects of Zn2+, Cd2+, Ca2+, and Mg2+

The effects of divalent cations (Zn2+, Cd2+, Ca2+, Mg2+) on the cytosol androgen receptor were determined by sedimentation into sucrose gradients. At low ionic strength (25 mM KCl, 50 mM Tris, pH 7.4), Zn2+ (200 microM total, which calculates to 130 nM free Zn2+ in 10 mM mercaptoethanol) causes a shift in the sedimentation coefficient of the rat Dunning prostate tumor (R3327H) cytosol receptor and rat ventral prostate cytosol receptor from 7.5 +/- 0.3 S to 8.6 +/- 0.3 S. Zn2+ stabilizes the 8.6 S receptor form in salt concentrations up to 0.15 M KCl in 50 mM Tris, pH 7.2. In low ionic strength gradients containing Ca2+ (greater than or equal to 200 microM) or Mg2+ (greater than or equal to 1 mM), the receptor sediments as 4.7 +/- 0.3 S. The dissociating effects of Ca2+ and Mg2+ can be fully reversed by sedimentation into gradients containing Zn2+ (200 microM total) or Cd2+ (10 microM total). In the presence of Zn2+ (200 microM total), Ca2+ (10 microM to 3 mM) converts the receptor to an intermediate form with sedimentation coefficient 6.2 +/- 0.2 S, Stokes radius 73 A, and apparent Mr approximately 203,000. The potentiating effect of Zn2+ on formation of the 8.6 S receptor (in the absence of Ca2+) and the 6.2 S receptor (in the presence of Ca2+) requires both the 4.5 S receptor and the 8 S androgen receptor-promoting factor. Sodium molybdate stabilizes the untransformed cytosol receptor but, unlike Zn2+, does not promote reconstitution of the 8.6 S receptor from its partially purified components. These results indicate that divalent cations alter the molecular size of the androgen receptor in vitro and thus may have a role in altering the state of transformation of the receptor.     

Binding of methyltrienolone to various androgen-dependent and androgen-responsive tissues in four animal species.

Comparative sucrose gradient studies of the in vitro binding of dihydrotestosterone (DHT) and of a synthetic androgen, methyltrienolone (R 1881), have been done with the cytosols of various tissues of the rat, mouse, cock and man. With rat prostate cytosol, the amount of R 1881 and DHT binding in the 8-9S region of the gradient was found to be comparable. Specific 8-9S peaks of R 1881 were also found in rat levator ani/bulbocavernosus and skeletal muscles and in the mouse kidney. Only 4-5S peaks could be demonstrated in the cock's comb while DHT under the same conditions showed both 8-9S and 4-5S binding. Binding of R 1881 to the cytosol of the hyperplastic prostate was polydispersed, and showed evidence of the presence of aggregates. Evidence was also found that R 1881 could bind to the progesterone receptor in rat uterus. Our study supports the theory that in a given species the androgen receptors are similar if not identical in all the tissues. The synthetic androgen R 1881 appears to be a useful tool for androgen receptor studies in various animal species provided that the tissue under study contains no progesterone receptor.     

Estradiol-17β receptors in the immature rat ovary

Estradiol binding components in the cytosol and nuclear fractions of the ovary from immature rats (22–28 days old) were characterized by $\text{in vitro}$ methods. Several of the biochemical characteristics of the estradiol binding components in the ovarian tissue were compared with the estradiol receptor from the uterus. The results suggest that the ovarian estradiol binding components are similar to the specific high affinity estradiol receptors in the uterus. In the cytosol of intact rat ovary a significant fraction of the total binding sites was found to be occupied, presumably by the endogenous estrogen. Following hypophysectomy there was a significant increase in the available cytosol binding sites. Evidence for translocation of cytosol receptor-estrogen (RE) complex to the nucleus was obtained for the ovary. The sedimentation properties of the RE complex of the ovary and the uterus are similar. The ovarian cytosol RE complex sediments at 7-8S in glycerol gradients at low ionic strength and at 4S in sucrose gradients at high ionic strength. Following extraction with 0.4 M KCl the ovarain nuclear RE complex sediments at 5S in sucrose gradients which is identical to that of the uterine nuclear receptor.     

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.