Biochem. J. (1980) 192,41-47 41Printed in Great Britain

Nuclear acceptor sites for androgen-receptor complexes in seminal-vesicleepithelium

Mitchell J. WEINBERGER and Carlo M. VENEZIALEDepartment of Cell Biology, Mayo Clinic and Graduate School ofMedicine, Rochester, MN 55901, U.S.A.

(Received 30 January 1980/Accepted 18 June 1980)

An assay method in vitro was developed and applied to quantify acceptor binding ofsteroid-receptor complexes in nuclei from isolated epithelium of guinea-pig seminalvesicle. Steroid-receptor complex prepared from 1-day-castrated animals was incubatedwith purified nuclei from 1-28-day-castrated animals in a medium containing0.15M-KCl. Free and bound steroid-receptor complexes were measured and the datawere submitted to Scatchard analysis. With nuclei from 1-day-castrated animals the Kdfor binding of cytosolic [3Hldihydrotestosterone-receptor complexes was found to be0.83 x 10-10M and the capacity for binding was 0.35 pmol/mg of nuclear DNA.Scatchard analysis consistently disclosed only a single line of constant slope and gavethe same kinetic constants for nuclei obtained from animals castrated up to 28 daysbefore assay. Administration of 2 mg of dihydrotestosterone, 3 a-androstanediol orandrosterone or 100,g of oestradiol-17f 1h before killing of the 1-day-castratedanimals that provided the nuclei resulted in a significant decrease in nuclear acceptorbinding of the steroid-receptor complex compared with untreated animals. Thus ourassay method disclosed nuclear acceptor sites that may be involved in responses toandrogens (and oestrogens) in vivo. We conclude that there is a class of nuclear acceptorsites of high affinity and limited capacity that may be occupied by steroid-receptorcomplexes in vivo.

The general concept has evolved that classes of'acceptor sites' exist in nuclei of steroid-hormone-responsive cells and that these 'acceptor sites' arepresumed to be responsible for determining thespecificity of interaction between steroid hormone-receptor complexes and DNA. The precise structureand function of these acceptor sites remain undocu-mented, but some studies performed in a variety ofsystems suggest that some subfraction of non-histone nuclear proteins is responsible for the'acceptor' activity (Buller & O'Malley, 1976).Substantial progress in characterizing nuclearacceptors has been made by Spelsberg and hisco-workers (Spelsberg et al., 1977) using the chickoviduct progesterone receptor system as a model.Others have studied, in less detail, nuclear acceptorsfor rat uterine oestrogen receptor (Higgins et al.,1973; Puca et al., 1975) and for prostatic androgenreceptors (Mainwaring et al., 1976; Klyzsejko-Stefanowicz et al., 1976). In the study of theforegoing model systems nuclei were purified fromtissues that were composed of heterogeneous celltypes. We have used, in contrast, an isolatedepithelial preparation from the guinea-pig seminal

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vesicle that is apparently cellularly homogeneous(Veneziale et al., 1974). Thus our nuclear pre-parations derive from a single cell type. Theadvantages in using such a tissue as a model systemfor the study of steroid-hormone action have beenoutlined (Veneziale, 1977; Veneziale et al., 1977b).

In the present paper we report the development ofan assay method in vitro for nuclear acceptor sitesthat appear to interact with androgen-receptorcomplexes in vivo. Regulation by androgen ofnuclear acceptor capacity per mg of DNA in theseminal-vesicle epithelium was also studied.

Materials and methods

Materials[1,2,4,5,6,7-3HlDihydrotestosterone (100-130 Ci/

mmol) and [ 1,2,6,7-3H]testosterone (80-100 Ci/mmol) were obtained from Amersham Corp.,Arlington Heights, IL, U.S.A. Aquasol was ob-tained from New England Nuclear Corp., Boston,MA, U.S.A. Non-radioactive steroids were obtainedfrom Steraloids, Wilton, NH, U.S.A. Bio-Gel P-10(50-100 mesh) was purchased from Bio-Rad Labor-

0306-3283/80/100041-07$01.50/1 (© 1980 The Biochemical Society

M. J. Weinberger and C. M. Veneziale

atories, Richmond, CA, U.S.A. Bovine serumalbumin and calf thymus DNA were purchased fromSigma Chemical Co., St. Louis, MO, U.S.A. Allreagents were of analytical grade.

AnimalsSexually active male guinea pigs (body wt.

600-800g) of the Mayo inbred strain were utilized inthis study. The animals were separated from theirharems and within several days scrotal castrationswere performed under diethyl ether anaesthesia.

Preparation ofcytosolicfractionsCytosolic fractions used in all experiments were

prepared from the tissues of 1-day-castratedanimals. Methods for the isolation of tissues fromcastrated guinea pigs have been previously described(Burns et al., 1979). Tissues were homogenized byusing a Potter-Elvehjem homogenizer at 0°C (12strokes) in 2 vol. (w/v) of TEDG buffer, pH 7.4[10 mM-Tris/HCl /1 mM-EDTA (disodium salt) /1 mM-dithiothreitol/10% (v/v) glycerol]. The homo-genate was centrifuged at 105 OOOg for 1 h at 20C inan SW 50.1 rotor in a Beckman L5-50 ultra-centrifuge. The clear supernatant fraction was

transferred to a clean tube and was retained in icefor subsequent use in nuclear acceptor studies (seebelow) or was fractionated by precipitation with40%-satd. (NH4)2SO4 as described elsewhere (Burnset al., 1979). The pellet was resuspended in TEDGbuffer with or without KCl in the original volume ofcytosol. For experiments requiring cytosolic recep-tors labelled with [3H]dihydrotestosterone, the initialhomogenization of tissues was performed in TEDGbuffer containing a saturating concentration of[3H]dihydrotestosterone (20 nM). [3H]Testosterone-receptor complex could not be prepared in thismanner because of substantial metabolism to [3HI-dihydrotestosterone. This was overcome by additionof a saturating concentration of [3H]testosterone(30nM) to the 105 OOOg supernatant fraction. Afterincubation for h at 0°C the [3H]steroid-receptorcomplex was precipitated by addition of (NH4)2SO4as described above. Dichloromethane/ethanol (7 :3,v/v) extracts of bound radioactivity (see below) inthe cytosolic and nuclear fractions were analysed byt.l.c. on Baker Flex lB silica-gel sheets (Steer &Veneziale, 1977). The solvent system used was

chloroform/ethyl acetate (4: 1, v/v). In each case the[3H]steroid initially added to the cytosol remainedunmetabolized.

Purified nuclearfractionsNuclei from tissues of animals castrated for

varying periods of time were purified by cen-

trifugation through 2.2 M-sucrose (Buchi &Veneziale, 1977). The purified nuclear fraction was

resuspended in 10 vol. (v/v) of TSKM buffer, pH 7.4

(0.25 M-sucrose/25 mm- KCl/lOmM-MgCl2/5OmM-Tris/HCl), and collected by centrifugation at 1500gfor 5 min at 40C in the TH-4 rotor in a BeckmanTJ-6 centrifuge. The washed nuclear material wasresuspended in fresh TSKM buffer at a concen-tration of 5 x 107-6 x 107nuclei/ml.

Nuclear acceptor assayPortions (50,ul) of the purified nuclear suspension

containing 25-50,ug of DNA and portions (12.5-500,U1) of the cytosolic fractions were co-incubatedin 3.0ml conical glass centrifuge tubes and wereadjusted to a final volume of 1.0ml by the additionof TEDG buffer with or without 0.15M-KCI. Incu-bation conditions were varied as described in theResults section to optimize the assay. Non-specificbinding of free radioactive hormone to nuclei wasdetermined by incubating nuclei with [3H]dihydro-testosterone at the same concentration as in thecytosolic fraction. At the end of each incubation, thenuclei were pelleted by centrifugation at 15OOg for5min at 4°C in a TH-4 rotor in the Beckman TJ-6centrifuge. The supernatant fraction was recoveredand saved for analysis of unbound hormone-receptor complex (see below). The nuclear pellet waswashed once with 2 ml of TEDG buffer and oncewith TE buffer [10 mM-Tris/HCl/1 mM-EDTA(disodium salt), pH 7.4] with or without 0.15 M-KCl.Bound radioactivity in the final washed nuclearpellet was extracted overnight with 1 ml of ethanol.The radioactivity of the ethanol extract was countedin 10.0 ml of Aquasol at 30% efficiency in aNuclear-Chicago mark II liquid-scintillationspectrometer.Unbound steroid-receptor complexes in

supernatant fractions of each assay mixture wereseparated from free steroid by gel filtrationessentially as described by Eisenfeld et al. (1976) oncolumns (1cmx lOcm) of Bio-Gel P-10 (equili-brated with TE buffer containing 0.15M-KCI) asfollows: 100,ul of 1% (w/v) Blue Dextran 2000 inTE buffer containing 0.15 M-KCl was added to eachsample (300,ul) and the resultant mixture waschromatographed. Columns were eluted with TEbuffer containing 0.15 M-KCI. The Blue Dextran-containing fractions, which also contained thesteroid-receptor complex, were collected in glassscintillation vials and their radioactivities counted inlOml of Aquasol.

Sucrose-density-gradient analysisIn some experiments the supernatant fractions

containing unbound hormone-receptor complexeswere subjected to sucrose-density-gradient analysiswith linear 5-20% (w/v) sucrose gradients preparedin TEDG buffer (Burns et al., 1979). Nuclear-bound hormone-receptor complexes were alsoanalysed on sucrose density gradients as follows:

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Nuclear acceptor sites in seminal-vesicle epithelium

after the nuclear pellets were washed with TE buffer(as above), radioactivity was extracted with TEDGbuffer containing 0.4M-KCI for Ih at 0°C. Aportion of this salt extract was applied to linear5-20% (w/v) sucrose gradients prepared in TEDGbuffer containing 0.4 M-KCI (Bums et al., 1979).

Injections in vivoIn certain experiments animals were injected

intraperitoneally with various steroids dissolved inpropylene glycol (0.4ml). Injections were given 1hbefore removal of tissues.

Chemical analysisProtein was quantified by the Lowry method as

modified by Ross & Schatz (1973) for samplescontaining thiol reagents. Crystalline bovine serumalbumin was used as standard. The 105 OOOg cytosolcontained about 20mg of protein/ml, and theresuspended (NH4)2SO4 precipitate contained about4mg of protein/ml. DNA was determined by theDische (1930) diphenylamine assay with calfthymus DNA as standard. Purified nuclear pre-parations (1-day-, 2-day- and 8-day-castratedanimals) from 1 g of tissue contained about 2-3 mgof DNA, which represented recovery of 40-50% ofthe DNA present in the tissue homogenate. Purifiednuclear preparations from 28-day-castrated animalscontained about 1 mg of DNA/g of tissue, repre-senting a recovery of only 20-25% of the DNApresent in the tissue homogenate.

ResultsBinding of cytosolic [3Hldihydrotestosterone-

receptor complexes to nuclei from the tissues of

1-day-castrated animals as a function of time at twodifferent temperatures is shown in Fig. 1. At 0°C thebinding was characterized by a gradual increasethroughout the 2h of incubation. At 250C, thebinding increased rapidly and then decreased gradu-ally to that observed after 2h of incubation at 0°C.At 250C cytosol [3H]dihydrotestosterone-receptorcomplexes undergo rapid dissociation. After lh at250C only 40% of the initial cytosol bindingremained (results not shown). In contrast, dissoci-ation at 0°C is negligible during this time. Theinstability of [3Hldihydrotestosterone-receptor com-plexes at 25 0C may explain the rapid fluctuations innuclear binding observed in Fig. 1.

Sucrose-density-gradient analysis of hormone-receptor complexes not bound after incubation withnuclei at 0°C for 2h demonstrated a peak ofradioactivity with a sedimentation coefficient of 7Sin the absence of salt. This is similar to a previouslyreported value for cytosol [3Hldihydrotestosterone-receptor complexes in seminal-vesicle epithelium(Burns et al., 1979), and suggests that the cytosolreceptor was maintained in its native form under theconditions of incubation (0°C) used in this assay.KCI (0.4M) extracted only 60% of the radio-

activity that could be extracted by ethanol fromnuclei previously incubated with cytosol [3HIdi-hydrotestosterone-receptor. Sucrose-density-gradi-ent analysis of the 0.4M-KCI nuclear extract (ingradients containing 0.4 M-KCI) demonstrated apeak of radioactivity sedimenting at 3-4S. Similarresults have been observed in previous studies inwhich [3Hldihydrotestosterone was incubated withintact seminal-vesicle epithelium preparations in

E 10

o 80

6

040

x

0 o 30 60 90 120Incubation time (min)

Fig. 1. Nuclear binding of cytosol [3Hldihydrotestoster-one-receptor

Portions (200,ul) of cytosol in TEDG buffer andpurified nuclear fractions (50,ul) from the tissues of1-day-castrated guinea pigs were incubated induplicate for various times at 0 and 25°C inTEDG buffer without added KCI. Nuclear bindingwas determined as described in the Materials andmethods section. 0, Incubations at 0°C; 0,incubations at 25°C.

-6

0la

c)0

.0.0

cd0

x

00 30 60 90 120

Incubation time (min)Fig. 2. Nuclear binding of partially purified [3HIdi-

hydrotestosterone-receptor complexPortions (250,p1) of crude or partially purifiedcytosol in TEDG buffer and nuclear fractions (50pl)from the tissues of 1-day-castrated guinea pigs wereincubated in duplicate for various times at 0°C inTEDG buffer without added KCI. Nuclear bindingwas determined as described in the Materials andmethods section. 0, Cytosol [3H]dihydrotestoster-one-receptor complexes; 0, partially purified [3H]-dihydrotestosterone-receptor complexes.

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M. J. Weinberger and C. M. Veneziale

vitro (Burns et al., 1979). Thus the sedimentationproperties of [3Hldihydrotestosterone-receptor com-plexes bound to purified nuclei in both cell-free andwhole-tissue incubations were similar.

The results shown in Fig. 1 suggested that theremay have been a temperature-dependent activationof hormone-receptor complexes resulting in a morerapid rate of nuclear binding at 250C than at 0°C.Since incubation at 25 C appeared to be deleteriousto the hormone-receptor complex and possibly tonuclei as well (Pikler et al., 1976), other ways ofeffecting receptor activation were considered. It hadbeen shown that (NH4)2SO4 precipitation was aneffective means of promoting activation of a varietyof steroid-hormone-receptor complexes (Buller &O'Malley, 1976). Most (60%) of the initial cytosol[3H]dihydrotestosterone-receptor complexes arerecovered after (NH4)2SO4 precipitation, but only20% of the initial cytosol protein was recovered.Thus, in addition to activation, partial purification ofreceptor was achieved. In Fig. 2 the binding ofcytosol and partially purified [3H]dihydrotestoster-one-receptor complexes to nuclei with time is

_

60-

'2

._0

.0

Cd

x

0

0

presented. The rate of binding to nuclei at 0°C wasgreater when partially purified receptor was used.After 1h of incubation, binding was nearly maxi-mal.The essential nature of the receptor for promoting

hormone binding to nuclei is demonstrated in Fig. 3.Substantial binding of radioactivity to nuclei wasobserved only after incubation with an activereceptor preparation. Partially purified receptor thathad been heat-treated (10min at 500C) and guinea-pig plasma, each containing the same amount of[3Hldihydrotestosterone as the active cytosolicfraction, were unable to promote very much bindingof radioactivity to nuclei.

The effect of increasing the concentrations ofpartially purified [3H]dihydrotestosterone-receptorcomplex on binding to nuclei in the absence andpresence of 0.15M-KCI is shown in Fig. 4. In theabsence of KCI, binding to nuclei was strictlyproportional to the concentration of partiallypurified receptor. When 0.15 M-KCl was included inthe incubation mixture, saturation of nuclearacceptor sites became apparent. We found that thebinding of [3Hldihydrotestosterone to the receptorand the sedimentation behaviour of the complexwere unaffected by 0.15 M-KCl. Therefore theappearance of saturable nuclear binding wasprobably not due to quantitative or qualitativechanges in the hormone-receptor complex inducedby KCl.

15f

0

100 200

Soluble fraction added (ul)300

Fig. 3. Receptor requirementfor nuclear bindingPartially purified cytosol in TEDG buffer containing0.15 M-KCI was heat-treated at 500C for 10min.Insoluble material was removed by low-speedcentrifugation. Guinea-pig serum was diluted (1: 9,v/v) with TEDG buffer containing 0.15 M-KCI, and[3Hldihydrotestosterone was added to give the samefinal concentration as in the partially purified cytosolfractions. Various amounts of the labelled solublefractions were incubated in duplicate at 0°C withnuclei (50,ul) purified from the tissues of 1-day-castrated guinea pigs in TEDG buffer containing0.15 M-KCI. Nuclear binding was determined asdescribed in the Materials and methods section. *,Partially purified [3H]dihydrotestosterone-receptorcomplexes; 0, heat-treated [3H]dihydrotestoster-one-receptor complexes; *, guinea-pig serum.

-

0

.0

0

-6

._

C)

0

CdI._

xIn

0I

0 100 200 300

Steroid-receptor complex added (pl)

Fig. 4. Effect of KCI on nuclear binding of partiallypurified [3H]dihydrotestosterone-receptor complexPartially purified cytosol in TEDG buffer with orwithout 0.15 M-KCI was incubated in duplicate for1 h at 0°C with nuclei (50,1) from tissues of1-day-castrated guinea pigs in TEDG buffer with orwithout 0.15 M-KCI. Nuclear binding wasdetermined as described in the Materials andmethods section. 0, No KCI; 0, + 0.15 M-KCI.

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Nuclear acceptor sites in seminal-vesicle epithelium

A representative saturation curve and its corre-sponding Scatchard (1949) plot are shown in Fig. 5.The results, as did those from numerous experi-ments, indicated the presence of one class of bindingsites with a Kd of 0.4 x 10-10M and a nuclearacceptor capacity (ne) of 0.29pmol/mg of DNA.

0

.

.>.

C 1o v.

cxS -.

xm

0

0.2

0

S.

e 0.1

-o100

300

Steroid-receptor complex added (ul)

When the data were plotted in the form of aLineweaver-Burk plot (not shown), the kineticparameters were found to be: Kd, 0.38 x 10-10m; n.,0.27 pmol/mg of DNA. Thus the two graphicalmethods gave excellent agreement. The Kd observedfor binding of the [3H]dihydrotestosterone-receptorcomplex to nuclear acceptor sites is about 10-foldlower than that observed for [3H]dihydrotesto-sterone binding to the receptor (Bums et al., 1979).To assess the physiological significance of nuclear

binding as observed by our assay, we determined theacceptor capacity for [3H]dihydrotestosterone-receptor complexes in nuclei after administration invivo of different hormones. As shown in Table 1,

Table 1. Effect of steroid administration on nuclearacceptor capacity

Nuclear acceptor assays were carried out as describedin the Materials and methods section in the presenceof 0.15M-KCI for 1 h at 00C. Nuclear and partiallypurified receptor fractions were prepared from thetissues of animals castrated 24 h before they werekilled. Animals used for the preparation of nucleiwere injected with the indicated steroids as describedin the Materials and methods section. Controlsreceived the vehicle alone. Binding data were analysedby the method of Scatchard (1949), and results areexpressed as the means + S.D. for three independentexperiments. Data were analysed by using Student'st test. Significance: *P < 0.001 compared withthe control.

Steroid injected Dose

NoneDihydrotestosterone3 a-AndrostanediolAndrosteroneOestradiol2 4 6

Specifically bound (pM)

2mg2mg2mg100ug

Nuclear acceptor capacity(pmol/mg ofDNA)

0.35 + 0.030.11 +0.03*0.10+ 0.03*0.07+ 0.03*0.12+0.06*

Fig. 5. Saturation curve (a) and Scatchard analysis (b)of binding of partially purified [3H]dihydrotestoster-

one-receptor complex to nucleiPartially purified cytosol in TEDG buffer containing0.15 M-KCl was incubated in triplicate with nuclei(5O,ul) prepared from the tissues of 1-day-castratedguinea pigs for 1 h at 00C in TEDG buffercontaining 0.15 M-KCl. Non-specific binding wasdetermined as described in the Materials andmethods section. Unbound [3Hldihydrotestoster-one-receptor complexes were isolated by gel fil-tration on Bio-Gel P-10, and bound hormone wasdetermined as described in the Materials andmethods section. 0, Total binding; 0, specificbinding (difference between total and non-specificbinding); *, non-specific binding. (b) The Kddetermined from the slope of the line obtained bylinear regression analysis was 0.4 x 10-10M and theconcentration of binding sites from the x-interceptwas 7.8pM. The correlation coefficient was -0.882.

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Table 2. Influence of castration on nuclear acceptorcapacity

Purified nuclei were prepared from the tissues ofanimals castrated for the times indicated. Partiallypurified receptor was prepared from the tissues ofanimals castrated 24 h previously. Assays werecarried out as described in the Materials and methodssection in the presence of 0.15 M-KCI for 1 h at 00C.See the legend to Table 1 for the remaining detailsof data analysis.Time aftercastration(days)

128

28

Nuclear acceptorcapacity

(pmol/mg of DNA)0.35 + 0.030.30+0.120.34 + 0.160.51 +0.05

RangeKd ofKd(pM) (pM)83 50-13070 40-1105 1 20-7043 10-100

45

M. J. Weinberger and C. M. Veneziale

administration in vivo of dihydrotestosterone, 3a-androstanediol, androsterone and oestradiol- 17fresulted in a substantial decrease in nuclear acceptorcapacity. The binding affinity in each case was notsignificantly different from the uninjected controls(results not shown).By using the nuclear acceptor assay we have

examined the acceptor capacity of nuclei isolatedfrom the tissues of animals castrated for variouslengths of time (Table 2). When expressed per mg ofnuclear DNA, there was no significant change innuclear acceptor capacity with increasing time aftercastration.

In a preliminary study nuclear acceptor capacityfor PHitestosterone-receptor complexes was evalu-ated. Although the Kd is the same as that for[3H]dihydrotestosterone-receptor complexes, thenumber of sites was only 0.18pmol/mg of DNA.Attempts to study the possibility that [3H]testo-sterone-receptor complexes and [3H]dihydrotesto-sterone-receptor complexes are binding to uniqueacceptor sites have been unsuccessful. We have beenunable to show competition between radioactive andnon-radioactive hormone-receptor complexes whenincubated with nuclei in a sequential fashion.

Discussion

In the present study, assay conditions have beendefined that are appropriate for the measurement ofnuclear acceptor sites for androgen-receptor com-plexes. We have chosen the isolated seminal-vesicleepithelium as a model system for these studies, sinceit is cellularly homogeneous by electron microscopy(Veneziale et al., 1974) and is dependent onandrogens for maintenance of its structure andfunction (Veneziale, 1977; Veneziale et al.,1977a,b). Nuclei purified from a homogeneouspopulation of cells may exhibit a simplified profile ofnuclear proteins when compared with a hetero-geneous nuclear preparation, and thus permit a moreprecise identification of acceptors for androgen-receptor complexes.

Assays were conducted at 0°C with partiallypurified receptor preparations under conditions ofmoderate ionic strength (0.15M-KCI). Low temper-atures and partially purified receptors were utilizedto minimize dissociation and degradation of steroid-receptor complexes and to limit changes inchromatin structure. These are important consider-ations, since proteolysis of receptors may result indecreased ability to bind DNA or chromatin withoutconcomitant loss of steroid binding (Sala-Trepat &Vallet-Strouve, 1974). On the other hand, degra-dation of chromatin may expose additional acceptorsites not routinely expressed in vivo (Spelsberg etal., 1977), thus resulting in overestimation of nuclearacceptor site capacity. Use of partially purified

receptors further decreased the possibility ofchromatin damage (Pikler et al., 1976), and, byactivating the receptor, provided rapid bindingkinetics at 0°C. Non-specific protein binding thatmight mask acceptor sites was also limited by usinga partially purified receptor preparation in nuclearacceptor assays. In the presence of 0.15M-KCI,binding of progesterone-receptor complexes waslimited to the highest affinity class of acceptors inchick oviduct chromatin (Pikler et al., 1976). Wehave also observed that binding of [3Hldihydro-testosterone-receptor complexes to nuclei in thepresence of 0.15 M-KCI was saturable and limited toa single class of acceptor sites with high affinity andlimited capacity. Binding was dependent on activereceptor, since neither guinea-pig plasma norinactive preparations of [3Hldihydrotestosterone-receptor complexes promoted binding of [3HIdi-hydrotestosterone to nuclei. By using values of0.35pmol of sites/mg of nuclear DNA in 1-day-castrated animals (Table 2) and 6 pg of DNA/nucleus there were about 1300 binding sites/nucleus.In our system the Kd for binding [3Hldihydro-testosterone-receptor complexes (0.8 x 10-10M) is10-fold lower than that reported for rat prostatenuclei, and the number of binding sites is less thanthe 2000-6000 sites/nucleus reported by the sameinvestigator (Liao, 1977). The reasons for thesediscrepancies are not obvious, but they might besomehow related to our use of a nuclear preparationpurified from a homogeneous cell population.

The relevance of cell-free binding of steroid-receptor complexes to nuclei has been questioned inseveral studies that were unsuccessful in attempts toshow decreased binding of steroid-receptor com-plexes in vitro after the administration in vivo ofphysiologically maximal doses of hormone(Chamness et al., 1974). There are several reasonsfor such a result that have been outlined, whichinclude dissociation, reassociation, or both, of thecomplex during nuclear isolation and the possibilitythat the initial binding to acceptor sites in vivo isquickly followed by a shifting to other nuclear sites(nuclear 'processing') (Spelsberg et al., 1977). In ourstudies, administration in vivo of various hormonesdid decrease steroid-receptor complex binding tonuclei. Three different androgens, dihydrotestoster-one, 3a-androstanediol and androsterone, wereequally effective in this respect. From previousstudies (Burns et al., 1979) we know that di-hydrotestosterone and 3 a-androstanediol bothpositively influence the seminal-vesicle epithelium invivo and that the different patterns of response maysuggest that each is active in its own right.Androsterone was found to be only marginallyeffective when used at a dose 3-fold higher than thatused in the present experiments. The ability ofandrosterone to decrease nuclear acceptor binding to

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Nuclear acceptor sites in seminal-vesicle epithelium 47

the same extent as dihydrotestosterone and 3 a-androstanediol is therefore difficult to explain.Perhaps it behaves in our system as oestriol behavesin the rat uterus (Anderson et al., 1975), i.e it(androsterone) is capable of inducing initial receptortranslocation as effectively as more potent andro-gens but fails to promote long-term retention ofreceptor in the nucleus. Long-term nuclear retentionof oestrogen-receptor complexes is thought to benecessary for eliciting uterine growth (Anderson etal., 1975). The oestradiol-17/1-induced decrease innuclear acceptor binding may be related to thepotent inhibitory effect on seminal-vesicle epitheliumobserved after administration in vivo (Burns et -al.,1979) to intact animals. We had demonstrated, inthe same study, that oestradiol-17,1 decreased thebinding of dihydrotestosterone to the androgenreceptor, and it may be suggested by the presentresults that oestradiol- 17/B translocates the androgenreceptor into the nucleus. The translocation of therat uterine oestrogen receptor into the nucleus afterincubation in vitro with high concentrations of di-hydrotestosterone (1pM) (Ruh et al., 1975) and afteradministration in vivo of high doses (3-15 mg) of di-hydrotestosterone (Rochefort & Garcia, 1976) havealready been described. It is interesting to speculatethat in pharmacological doses oestrogen may act asan anti-androgen at the target-tissue level by theformation of a less than fully active androgenreceptor-hormone complex.

Regulation of target-cell sensitivity to steroidhormones may be at two levels. On one level,changes in cytoplasmic receptor content may makea tissue more or less responsive to the hormone. Onanother level, changes in nuclear acceptors may alterresponses both quantitatively and qualitatively.Cytoplasmic receptor concentration (binding/mg ofprotein) in rat prostate gland is regulated byandrogens shortly after castration, but becomesindependent of androgens after prolonged periodsafter castration (Sullivan & Strott, 1973). In theseminal-vesicle epithelium, which undergoes sub-stantial changes in morphology and function aftercastration (Veneziale, 1977; Veneziale et al., 1977a;Barham et al., 1979), we evaluated the nuclearacceptor capacity from 1 to 28 days after castrationto check whether similar patterns of regulationoccurred. We observed that the nuclear acceptorcapacity (per mg of DNA) and Kd remainedunchanged. This is not entirely unexpected, and isreally consistent with the fact that the tissuemaintains its ability to respond to androgens despitelong-term castration (Bums et al., 1979). However,qualitative changes in nuclear acceptors that mayhave occurred would not have been detected in thisassay.

Further studies in this area must await a detailedcharacterization of nuclear acceptor components,which can be most easily accomplished in acellularly homogeneous target tissue like the guinea-pig seminal-vesicle epithelium.

This work was supported by N.I.H. grant HD12657and by the Mayo Foundation.

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