Intracellular receptors for 5α-dihydrotestosterone 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

19

INTRACELLULAR RECEPTORS FOR 5a-DIHYDROTESTOSTERONE 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.

V. Hansson, 0. Djoseland, E. Reusch, A. Attramadal and 0. Torgersen Laboratory of Histochemistry, Institute of Pathology and Laboratory of Endocrinology, Medical Department B, Rikshospitalet, Oslo, Norway

Received: 4119173

ABSTRACT Epididymal cytosol fractions of adult short-time castrated rats contained at least two different androgen protein complexes by experiments --in vivo (Complex I and II). Complex I is probably located intracellularly in the epididymal cells. It was specific for 5o-dihydrotestosterone (DHT) and appeared to be very similar to the cytoplasmic DHT-receptor complexes in rat ventral prostate. By ultracentrifugation on sucrose gradients, it sedimiented 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) inothe testicular flui!. It was eluted between immunoglobulin G (IgG) (53 A) and albumin (36 A) by gel filtration on Sephadex G-200. The sedimentation coefficient was 4.5-5 S (mean 4.6 S,,2U) 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 theintraluminal 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.

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INTRODUCTION The epididymal cytosol fraction contains at least one specific DRT binding protein (ABP), initially thought to represent the cytosol receptor for DHT in .the epididymis (l-5).

This protein has, however,

later been detected both in the testis cytosol fraction and in the testicular efferent duct fluid (6-8), and ABP is now considered a transport protein for androgen in the testicular fluid.

In previous

publications the specific retention of androgen by epididymal nuclei after the administration of tritiated testosterone as well as DHT -in vivo was described (1,3,9). others (10,ll).

Similar observations have also been made by

Since nuclear binding of steroid hormones in target

tissues appears to be contingent upon the presence of a cytosol receptor, it seemed probable that also the epididymis should contain a soluble receptor for DHT in the cytoplasm. The purpose of the present study was to examine whether the epididymal cytosol fraction contained a specific binding protein for androgen in addition to ABP.

If so, it would be of interest to examine the

physicochemical properties and steroid specificity of this androgenprotein complex as well as to compare it with both ABP and the cytosol receptors for DHT in the ventral prostate.

MATERIALS AND METHODS Animals Adult male Wistar rats, weighing about 300 g, were used. A number of the animals were bilaterally orchidectomized 36 hrs prior to the In one experiment, 10 animals were unilaterally castrated experiments. 4 weeks prior to the experiments in order to empty the epididymis for its luminal contents. The remaining testis was removed 24 hrs before the sacrifice.

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Chemicals -1,2-3H-Testosterone (45 Ci/mMole) and 1,2-3H-5~-dihydrotestosterone (17B-hydroxy-5a-androstan-3-one; DHT; 44 Ci/mMole) were obtained from the New England Nuclear Corporation. The purity of DHT was more than 98 per cent at the time of the experiments; its radio-chemical homogeneity was checked by thin layer chromatography (TLC) (2). Trizma base, bovine serum albumin (BSA), p-chloro-mercuri-phenyl-sulphonate (PCMPS) and protease (bacterial type VII) were obtained from Sigma Chemical Co., human y-globulin (IgG) from A.B. Kabi (Sweden), Sephadex Cl00 and G-200 from Pharmacia (Sweden). Buffers The following buffers were used: 0.1 M Tris HCl buffer, pH 7.4 (Tris); 0.1 M Tris HCl pH 7.4, containing 0.5 M NaCl (0.5 M Tris Na); and 0.25 M sucrose in 0.01 M Tris containing 1 mM EDTA (TES) pH 7.4. Labeling of the tissues In the majority of cases animals in groups of 10 were injected with 100 PCi 3H-testosterone per animal and killed after 60 min. The organs were rapidly removed and homogenized in 3 volumes Tris or TES at O-2" C, using an Ultra Turrax (Model TP) homogenizer. The homogenates were centrifuged at 105,000 x g for 60 min at 2-4" C and the cytosol fractions stored at -20" C until analyzed. Stability of the DHT binding proteins In order to test the stability of the different DHT-protein complexes of the epididymal and ventral prostate cytosol fractions 20 animals castrated 36 hrs previously were injected with 100 UCi 3Htestosterone and killed 60 min later. Cytosol fractions were prepared as dlescribed above, divided into 2 ml aliquots and treated as follows: 1) incubation at 25O C for 30 min (control), 2) addition of 1 mM PCMPS and incubation at 25' C for 30 min, 3) addition of 500 ug protease and incubation at 25O C for 30 min, 4) incubation at 50" C for 30 min, 5) absorption overnight at 4" C with dry charcoal (1 mg Norit A/mg protein). After absorption, the charcoal was removed by centrifugation twice at 800 x g for 15 min and the absorbed cytosols were reincubated with 2 nM 3H-DHT for 30 min at 25O C. Binding of 3H-DHT in epididymal and prostate cytosol fractions was assayed by Sephadex G200 gel filtration. Gel filtration chromatography Columns of Sephadex G-100 (1.6 x 63 cm) and Sephadex G-200 (1.6 x 40 cm and 2.5 x 36 cm) were packed as recommended by the manufacturer (Pharmacia, Uppsala, Sweden). The columns were equilibrated with Tris and the flow rate was 10 ml/hr, downwards. The columns were calibrated with blue dextran, IgG and BSA. All gel filtrations were performed at 4" C. Aliquots were taken from each fraction for determinations of the radioactivity and the optical density at 280 nm.

22

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Sucrose gradient centrifugation Ultracentrifugation in sucrose gradients was performed according to Martin and Ames (12), using BSA and IgG as reference standards. Samples of 250 ~1 were layered on the top of 5-20 per cent (w/v) sucrose gradients prepared in Tris or in 0.5 M Tris Na buffer using a Buchler gradient apparatus. The gradients were centrifuged at O-4" C in a Beckman Spinco ultracentrifuge (Type L-50) using a SW 50 or a SW 56 rotor. After puncturing the tubes in the bottom, fractions of 10 droplets (130 ~1) each were collected. The linearity of the gradients was confirmed by means of a Zeiss refractometer. The sedimentation rate of unknown protein was calculated from the rate of sedimentation relative to that of BSA (4.6 S) and IgG (6.9 S) (13). Characterization of radioactive steroids Following gel filtration, protein-bound as well as free radioactivity were examined by thin layer chromatography (TLC) and the major metabolite bound in complex I was identified by crystallization to constant specific activity (2). Other analytical procedures The optical densities of the fractions obtained by gel filtration and the sucrose gradient centrifugation were recorded at 280 nm. Total protein was determined by the method of Lowry -et al. (14) with BSA as reference standard. Radioactivity was measured by liquid scintillation counting with an efficiency of about 44 per cent.

RESULTS As illustrated in Fig. 1, the epididymal cytosol fraction of adult, short-time castrated rats contained at least two different androgenprotein complexes.

Complex I (R) was excluded from Sephadex 6200;

whereas complex II (ABP) became eluted between IgG (53 ii> and albumin (36 1).

About half of the radioactivity in the cytosol fraction was

bound to proteins (complex I and II), whereas the rest was free and was eluted corresponding to materials of small molecular weight (Fig. 1) Since ABP has been shown to disappear rapidly after ligation of the efferent ducts or castration (6,8), 10 rats were unilaterally castrated 4 weeks prior to the experiments. 24 hrs before sacrifice.

The remaining testicle was removed

On the hemicastrated side, complex II was

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completely absent, and all the bound radioactivity became eluted in the void volume from a column of Sephadex G-200 (Fig. 2).

On the control

side (castrated 24 hrs), both peaks of radioactivity were found (Fig. 2) and the radioactivity bound to ABP was 3-4 times higher than that eluted in the void volume of Sephadex G-200 columns.

CPM 500 _ 400 _ 300 _ 200 _ 100 _ B# 10

20

30

40I

FRACTION

50I

60

NO

Figure 1 Gel filtration of an epididymal Tris cytosol fraction (1.5 ml) on a column of Sephadex 6200 (1.6 x 40 cm). Adult rats bilaterally castrated for 36 hrs were injected with 100 ?..NZi 3H-testosterone. The animals were sacrificed after 60 min and a 105,000 x g cytosol fraction prepared. Elution buffer: 0.i M Tris pH 7.4. Flow rate: 5 ml/h downwards. Temperature: 4" C. Fractions of 1 ml were collected. The elution volumes of blue dextran (Vo), y-globulin (IgG) and,albumin (Alb) are indicated by arrows. R: complex I. ARP: complex II.

Gel filtration of ventral prostate cytosol fractions, labeled in vivo under identical conditions, gave one single peak of protein--bound radioactivity; excluded both from Sephadex Cl00 6200

(not shown).

and Sephadex

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6

-

4 WEEKS CASTRATED

b---o

24 HOURS CASTRATED

FRACTION

NUMBER

Figure 2 Gel filtration chromatography on Sephadex G-200 (2.5 x 36 cm) of epididymal cytosol fractions (4 ml) obtained from animals which had been unilaterally castrated on the right side for 4 weeks and bilaterally castrated for 24 hrs. The animals were given 100 1.1Ci. 3H-testosterone in vivo and sacrificed 60 min after the injection. -U

4 weeks castrated side

w

24 hrs castrated side.

The arrows show the elution positions of blue dextran (Vo), IgG and albumin @lb). R: complex I. ABP: complex II.

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BSA $_

CPM 500

400

J

300 200 100

lb

;o

Fraction

No.

Figure 3 -Sucrose gradient centrifugation of fraction (250 ~1) prepared as described Centrifugation was performed for 18 hrs rotor. The arrows show the position of

an epididymal in the legend at 42,000 rpm albumin (Alb)

Tris cytosol to Fig. 1. using a SW 56 and IgG.

Sucrose gradient centrifugation of an epididymal cytosol fraction from adult, short-time castrated rats revealed one major peak of bound radioactivity, migrating with a sedimentation constant of 4.5 - 5 S (mean 4.6 S~O,~) (Fig. 3).

This peak of protein bound radioactivity

with sedimentation coefficient of 4.6 S disappeared completely from the castrated side after unilateral orchidectomy for 4 weeks, suggesting that the 4.6 S peak corresponds to the complex II observed by Sephadex G-200 gel filtration (Fig. 1).

Complex I was under these conditions

distributed all through the sucrose gradient and could by this method

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be demonstrated only after removal of complex II.

As illustrated in

Figure 4, the protein-bound radioactivity on the 4 weeks castrated side sedimented as heavy aggregates, 8-10 S and 3-4 S complexes.

However,

when the cytosol fractions were made 0.5 M in sodium chloride and centrifuged through S-20 per cent sucrose gradients containing the same concentration of salt, all of the protein-bound radioactivity migrated with a sedimentation constant of 3-4 S, and no radioactivity was found either at the bottom of the tubes or in the 8-10 S region (Fig. 4).

Hemicastrated Lweeks CPM 200

-

100 -

50

- 4

BOTTOM

001 M Tris+0.5

lb FRACTION

2b NO.

3b

M NaCL

TOP

Figure 4 Sucrose gradient analyses of epididymal Tris cytosol fractions (250 ~1) obtained from the 4 weeks castrated side of hemicastrated rats given 100 PCi 3H-testosterone by an intramuscular injection 60 min prior Centrifugation was performed for 18 hrs at 42,000 rpm, to the sacrifice. using a SW 56 rotor. The arrows show the position of IgG and BSA. The gradients were made either in 0.01 M Tris alone or containing 0.5 M NaCl.

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-Steroid specificity of complexes I and II In order to examine the steroids bound to complexes I and II following administration of 3li-testosterone in vivo,

both the protein-

bound and the free radioactivity from the 4 week and the 24 hr castrated sidlewere pooled, extracted with diethylether and subjected to TLC. Bound and free radioactivity were separated by Sephadex G-100.

On both

sides more than 90 per cent of the protein-bound radioactivity was isopolar with DHT (Fig. 5).

After addition of a non-radioactive carrier,

the isolated DHT bound in complex I was identified by crystallization to clonstant specific activity (Table 1). The free (non protein-bound) radioactivity after the gel filtration (fractions 40-55) consisted of a number of different steroids.

One

peak was found at the starting line accounting for 10-20 per cent of the radioactivity.

The largest peak, accounting for 20-50 per cent of

the radioactivity, corresponded to DHT (RF: - 0.52).

Smaller amounts of

radioactivity iso-polar with 5a-androstane-3a and @-17$-diol, testosterone, 4-androstene-3,17-dione

and 5a-androstane-3,17-dione were also

found to be present (Fig. 5). Properties receptor of the rat ventral prostate. Cytosol fractions of the epididymis and ventral prostate of 24 hr castrated rats were subjected to different treatments in order the stability of the androgen-protein complexes. by Sephadex G-200 gel filtration. results of these experiments.

to

test

Binding was assayed

Table 2 gives a summary of the

Epididymal complex I and the cytosol

receptor of the rat ventral prostate were both destroyed by heating at 50" C for 30 min, treatment with 1 mM PCMPS, and charcoal absorption.

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The binding of DHT to complex II was not or only slightly affected by such treatments (Table 2).

Without ABP bound

Without ABP free 4 3

- 94% DHT

? 2F r 1%

10 wDv?D

12

15cm

-umcm

5

10

15cm

--Qmam

345

12

With ABP bound

345 With ABP free

31*

-3 ‘;” -2 e z -1 v

5 ral~~rw~ 12345

10

15cm

5 10 aCaa7w aa= 12 345

15cm

Figure 5 TLC (methylene chloride/diethylether 4:1, v/v) of protein-bound (fractions 15-30) and free steroids (fractions 40-55) after gel filtration on Sephadex GlOO. Upper diagram: 4 week castrated side (without complex II). Lower diagram: 24 hr castrated side (both complex I and II). After the chromatography, the plates were divided into 0.5 cm segments and the radioactivity examined in each segment. 2) testosterone, 3) DHT, Standards: 1) *5a-androstane-3-176-diol, 4) 4-androstene-3,17-dione, 5) 5o,-androstane-3,17-dione. *In this system, the 3a and 3S epimers of 5o-androstane-3,1B-diol have the same mobility.

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Table 1 Recrystallization to constant specific activity of the isolated DHT bound in complex I.

Crystallization solvent

Weight of the crystals (mg)

CPM/mg

1.

Acetone/water

4.9

283

2.

Methanol/water

5.2

280

3.

Hexane/benzene

5.6

276

4.

Cyclohexane/ethylacetate

4.6

292

Table 2 Properties of epididymal complex I, complex II (ABP) and the cytosol receptors of the rat ventral prostate. Adult rats, 24 hr castrated rats were injected intramuscularly with 100 yCi 3H-testosterone/animal 60 min prior to the sacrifice.

Ventral prostate Sedimentation rate

Stokes radius

Complex I

4s 6-7 S 8-10 S heavy aggr.

4s 8-10 S heavy aggr.

excluded from G-200

excluded from G-200.

Complex II

4.5-5 sx

46-48 ix

Heating at 50° C for 30 min.x

+

1mM

+

PCMPS

500 ug protease Charcoal absorption

(-> (+) X

+

Binding activity completely destroyed Binding activity not reduced More data about complex II (ABP) is given in ref. 5 and 6.

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DISCUSSION The present results demonstrate that the epididymal cytosol fraction of adult, short-time castrated rats contains at least two androgenbinding proteins, both of which exhibit high affinity for DHT.

Although

these complexes could not be separated by ultracentrifugation on sucrose gradients, they were completely separated by Sephadex 6200.

One of

them (complex II) is identical to the testicular androgen binding protein (ABP) (6) which is secreted in efferent duct fluid and passes through the epididymis (7,8).

The other (complex I) appeared to be very

similar to the cytosol receptors of the rat ventral prostate.

At low

ionic strength, both epididymal complex I and the cytosol receptors for DHT in the rat ventral prostate easily formed aggregates when examined by sucrose gradient centrifugation.

These aggregates dissociated into

smaller complexes (3-4 S) at high ionic strength.

Complex I and pros-

tatic receptors also showed identical chromatographic behavior on Sephadex 6200,

similar steroid specificity and stability (Table 2).

The presence of complex I on the castrated side after unilateral orchidectomy for 4 weeks indicates that this components is not derived from the testis but is confined to the epididymis.

Furthermore, the similar-

ity between epididymal complex I and the prostatic receptors for DHT also suggests that complex I represents the cytoplasmic receptor for DHT within the epididymal cells. Complex I is probably identical to the 8.5 S and 4 S complexes which have been described in the epididymal cytosol fraction of hemicastrated rats (11).

Recent results of Blaquier

(15) also demonstrate that DHT-

protein complexes in epididymal cytosol of 4 weeks hemicastrated animals

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were bound by isolated ventral prostate cell nuclei in a specific manner. Such a 'biological cross reaction" indicates a high degree of structural similarity between cytoplasmic receptors in the prostate and epididymis. The similarity between the epididymal complex I and the androgen receptors in the rat ventral prostate reported here also suggests that the receptor proteins of these androgen dependent target organs belong to the same family of proteins. The changes in size depending on ionic strength are also a common feature for all steroid hormone receptors (16).

The biological impor-

tance of these aggregation phenomena is, however, not clear. Complex I corresponds to the slower moving peak observed by polyacrylamide gel electrophoresis

(PAGE) (4), and it has recently been

shown that this slower moving zone of protein bound radioactivity is maintained after 8 days of castration (17).

This is in contrast to ABP

which moves faster on PAGE and disappears rapidly after efferent duct ligation or castration (6-8, 18). To what extent complex I is identical with the androgen-protein complex extracted from epididymal nuclei has not been established.

There

is a general opinion, though the absolute proof is still lacking, that all steroid receptors extracted from the nuclei are of cytoplasmic origin (16). Recent studies of Tindall -et al. (19) h ave shown that nuclear uptake and binding to nuclear receptors in the epididymis are dependent on binding of DHT to the slower moving peak as demonstrated by PAGE and independent of binding to the faster moving peak (ABP).

Although cypro-

terone acetate greatly diminished cytoplasmic and nuclear uptake of androgen and completely abolished binding to the slower moving peak in

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the cytosol as well as the nuclear receptors, it had no effect on binding to ABP (17).

Furthermore, equal concentrations of this slower

moving complex were found in the caput and cauda epididymis (17), in contrast to ABP, which is mainly located in the caput epididymis (20).

ACKNOWLEDGEMENTS The authors wish to thank dr. odont. Stig Schultz-Haudt, Ph.D., for his interest and valuable help. Financial support was given by the Norwegian Research Council for Science and the Humanities and the Norwegian Cancer Society (Landsforeningen mot Kreft, Oslo).

REFERENCES 1.

Hansson, V., and Tveter, K.J., ACTA ENDOCR. (Kbh.) 66, 745 (1971).

2.

Hansson, V., and Djoseland, O., ACTA ENDOCR. (Kbh.) 71, 614 (1972).

3.

Hansson, V., Tveter, K.J., Unhjem, O., and Djoseland, O., J. STEROID BIOCHEM. 3, 427 (1972).

4.

Ritz&, E.M., Nayfeh, S.N., French, F.S., and Dobbins, M.C., ENDOCRINOLOGY 89, 143 (1971).

5.

Hansson, V., STEROIDS 20, 575 (1972).

6.

Hansson, V., Djoseland, O., Reusch, E., Attramadal, A., and Torgersen, O., STEROIDS 21, 457 (1973).

7.

French, F.S., and Ritz&,

8.

Ritz&, E.M., Dobbins, M.C., French, F.S., and Nayfeh, S.N., IV INT. CONGR. OF ENDOCRINOLOGY, Wash, D.C., 1972. Abstract, p. 79.

9.

Hansson, V., Djoseland, O., Tveter, K.J., Unhjem, O., Attramadal, A., and Torgersen, O., IV INT. CONGR. ENDOCRINOLOGY, Wash. D.C., 1972. Abstract, p. 19.

E.M., J. REPROD. FERTIL. 32, 479 (1973).

10.

Tindall, D.J., French, F.S., and Nayfeh, S.N., BIOCHEM. BIOPHYS. RES. COMM. 49, 1391 (1972).

11.

Blaquier, J.A., BIOCHEM. BIOPHYS. RES. COMM. 45, 1076 (1971).

12.

Martin, R.G., and Ames, B.N., J. BIOL. CHEM. 236, 1372 (1961).

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13.

Schultze, H.E., and Heremans, J.F., MOLECULAR BIOLOGY OF HUMAN PROTEINS WITH SPECIAL REFERENCE TO PLASMA PROTEINS, Elsevier Publishing Co., Amsterdam, vol. 1, 1966.

14.

Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J., J. BIOL. CHEM. 193, 265 (1951).

15.

Blaquier, J.A., and Calandra, R.S., ENDOCRINOLOGY, in press.

16.

Erdos, I., ADVANC. BIOSCI. 7, 400 (1971).

17.

Tindall, D.J., Hansson, V., Weddington, S.C., French, F.S., and Nayfeh, S.N., PROGRAM 55th ENDOCRINE SOC. MEETING, June 1973.

18.

French, F.S., and Ritz&,

19.

Tindall, D.J., Weddington, S.C., French, F.S., and Nayfeh, S.N., BIOCHEM. BIOPHYS. RES. COMM., in press.

20.

Hansson, V., Weddington, S.C., Tindall, D.J., French, F.S., Nayfeh, S.N., and Ritz&, E.M., PROGRAM SOC. FOR STUDY OF REPRODUCTION, August 1973.

E.M., ENDOCRINOLOGY, in press.