Urological Research © by Springer-Verlag 1977
Urological R e s e a r c h 5, 1 6 9 - 1 7 3 (1977)
Androgen Receptors in the Prostate of the Rhesus Monkey R. Ghanadian, G. Auf, C.B. Smith, G.D. Chisholm, and N.J. Blacklock 1 P r o s t a t e R e s e a r c h L a b o r a t o r y , U r o l o g i c a l Unit, Royal P o s t g r a d u a t e Hospital, London, and 1Royal Naval Hospital, Haslar, U.K.
Received:
M e d ic a l School, H a r n r n e r s r n i t h
July 29, 1977
S u m m a r y . T h e p r e s e n c e of a n a n d r o g e n r e c e p t o r p r o t e i n i n the s u p e r n a t a n t p r e p a r a t i o n of the p r o s t a t e f r o m R h e s u s m o n k e y ( m a r a c a r n u l a t t a ) i s d e s c r i b e d . T h e m o l e c u l a r w e i g h t of t h i s r e c e p t o r p r o t e i n w a s f o u n d to be 2 . 8 - 2 . 9 x 1 0 5 d a l t o n s . T h e l e v e l s of f r e e a n d b o u n d a n d r o g e n r e c e p t o r s w e r e m e a s u r e d i n the c a u d a l a n d c r a n i a l l o b e s of the p r o s t a t e b y a n e x c h a n g e a s s a y u s i n g r n e t h y l t r i e n o l o n e (R1881). T h e c o n c e n t r a t i o n of the f r e e b i n d i n g s i t e s i n the c a u d a l l o b e r a n g e d b e t w e e n 3 . 7 - 2 3 . 7 f m o l / r n g p r o t e i n . T h e v a l u e i n the c r a n i a l l o b e w a s 2 . 0 - 7 . 7 f m o l / r n g p r o t e i n . T h e t o t a l b i n d i n g s i t e s i n the c a u d a l l o b e r a n g e d b e t w e e n 3 6 . 0 - 1 1 2 . 7 a n d i n the c r a n i a l b e t w e e n 2 1 . 2 - 5 5 . 0 f m o l / m g . T h e b o u n d r e c e p t o r r a n g e d between 43.3-109.0 and 19.1-47.3 fmol/rn~ protein for caudal and cranial lobes respectively. The l e v e l of b o t h b o u n d a n d f r e e r e c e p t o r s w a s f o u n d to b e s i g n i f i c a n t l y h i g h e r i n the c a u d a l l o b e . T h i s d a t a s u g g e s t s t h a t the two l o b e s of the p r o s t a t e i n the R h e s u s m o n k e y c a n b e e q u a t e d w i t h t h e two z o n e s of the h u m a n p r o s t a t e i n r e s p e c t of a n d r o g e n r e s p o n s i v e n e s s . Key words: Androgen receptors
- Rhesus monkey - Prostate
T h e e x i s t e n c e of a r e c e p t o r p r o t e i n w h i c h b i n d s s p e c i f i c a l l y to d i h y d r o t e s t o s t e r o n e h a s b e e n d e m o n s t r a t e d i n the p r o s t a t e of s e v e r a l s p e c i e s (3). In a p r e l i m i n a r y s t u d y f r o m t h i s l a b o r a t o r y it w a s d e m o n s t r a t e d t h a t the c a u d a l a n d c r a n i a l l o b e s of the p r o s t a t e i n the R h e s u s m o n k e y ( m a r a c a m u l a t t a ) w e r e a b l e to t a k e up a n d r e t a i n a n d r o g e n a n d t h a t the u p t a k e of t e s t o s t e r one b y the c a u d a l l o b e of the p r o s t a t e w a s significantly higher than any other target or n o n - t a r g e t t i s s u e (4). B o t h i n the r e l a t i o n s h i p to s u r r o u n d i n g s t r u c t u r e s a n d i n t h e i r d i f f e r e n t i al h i s t o l o g i c a l c h a r a c t e r i s t i c s the c r a n i a l lobe of the p r o s t a t e of the m o n k e y a p p e a r s to b e h o m o l o g o u s w i t h the c e n t r a l z o n e of the h u m a n a n d the c a u d a l l o b e w i t h the p e r i p h e r a l z o n e (2). T h e p r e s e n t s t u d y w a s c a r r i e d out to c h a r a c t e r i s e a n d r o g e n r e c e p t o r s i n the c y t o s o l of R h e s u s m o n k e y p r o s t a t e a n d to d e t e r m i n e the c o n c e n t r a t i o n of t h e s e r e c e p t o r s i n the cranial and caudal lobes.
M A T E t ~ I A L S AND M E T H O D S Monkeys Rhesus monkeys (maraca mulatta) aged between 2 a n d 10 y e a r s , w e r e u s e d i n t h i s s t u d y .
- Receptor assay.
Prostate and other tissues w e r e dissected out whilst the animals w e r e under general anaesthesia. S a m p l e s w e r e kept in containers on ice and transported to the laboratory. All the operative procedures w e r e p e r f o r m e d between ii and 15 h.
Steroids (i~, 2~, 3H) 5a-dihydrostestosterone (3H)DHT (specific activity 58 Ci/mrnol; Radiochemical Centre, Arnersham, U.K.). Unlabelled 5~dihydrotestosterone (DHT) (Sigma Chemicals). (6,7-3H) rnethyltrienolone (3H) R1881 (specific activity 55.5 Ci/rnmol) and unlabelled R1881 were kindly supplied by Roussel Uclaf, France.
Buffers
Buffer A: Tris buffer (20 rnM) containing 1.5 mM EDTA and 2 rnM rnereaptoethanol adjusted to pH 7.4. Buffer B: Tris buffer (i0 rnM) containing 1.5 rnM EDTA and 250 rnM sucrose adjusted to pH 7.4. Both buffers were freshly prepared and stored at 4°C
170
Urol.
Two solutions (i and 2) of dextran coated charcoal were prepared in buffer B. Solution 1 consisted of 5 % water washed Norit GSX charcoal (Hopkins and Williams) and 0.5% dextran T-70 (Pharmacia). Solution 2 consisted of 1.25% water washed Norit GSX charcoal and 0.625 % dextran T-70. Both solutions were stored at 4°C.
Solvents Ethanol, Methanol, Toluene and Triton X-100 were all of Analar grade (British D r u g Houses).
DNase and RNase from bovine pancreas and pronase from streptomyces griseus (Sigma Chemicals). Column
Chromatography
Sepharose
6B
(Pharmacia
Fine
Chemicals).
Scintillation Fluid Toluene: Triton X-100 (2 : i) containing 0.4% 2,5-diphenyloxaZole (Koch-Light Laboratories). Preparation
of Cytosol
Sections of either caudal or cranial lobes of the prostate were sliced, minced, blotted with filter paper and weighed. The minced tissue was placed in a pre-cooled B24 tube and homogenised in buffer (1:6 w/v). Homogenisationwas carried out using an Ultra Turrax type TPI8-10 (Junks and Kunkle. AG) using 5 strokes each of I0 seconds with 30 seconds cooling intervals. The homogenate was then centrifuged at 105,000 g for 1 h at 4 ° C. The resulting supernatant (cytosol fraction) was cleanly pipetted taking special care to avoid contamination from the lipid layer on the top. All the above procedures were carried out at 0-4 °C. Free endogenous hormone was removed from the cytosol fraction by incubating the cytosol with charcoal buffer (solution i) in a ratio of i0:i v/v at 0-4 °C for 10 rains. The steroid bound to charcoal was precipitated by centrifugation at 3700 g at 4 ° C for 20 rains. Aliquots of i00 ~i of the cytosol were then used for protein estimations (8). The remaining cytosol was used for the characterisation and the assay of both unoccupied and total binding sites.
Characterisation of Receptor Aliquots of the cytosol stripped of endogenous h o r m o n e were incubated with 50 n M of (3H) dihydrotestosterone in the presence and absence of unlabelled dihydrotestosterone at 15 ° C for 16 h. Unbound steroids were r e m o v e d by incubating the cytosol with dextran charcoal solution 2 (2:1 v/v) at 0-4 ° C for I0 rains. Fol-
Vol.
5, No.
4 (1977)
lowing centrifugation at 1000 g for 15 mins the supernatant was eluted on a precalibrated column (55 cmx 2.5 cm) of Sepharose 6B (7) at a flow rate of 40 ml/h with buffer A. Fractions (4 ml) were collected in an LKB 7000 fraction collector. Aliquots (2 ml) of each fraction were transferred to scintillation vials and scintillation fluid (15 ml) was added. The radioactivity was counted in a scintillation spectrometer. The results were expressed as counts per minute per fraction. Measurement
Enzymes
Res.,
of the Receptors
a) Free Binding Sites. Unoccupied binding sites (free receptors) were measured in the cytosol fraction as follows: For triplicate assay 6 test tubes were set up, each pair containing 0.i pmol (3H) R1881 with and without 500 prnol radioinert R1881 in ethanol. The solutions were evaporated to dryness and subsequently transferred to crushed ice. Aliquots of 0.1 ml cytosol were added to each tube and incubated at 0-4o C for 1 h. Unbound R1881 was removed by adding 50 ~i dextran charoal (solution 2) which was then vortexed and incubated at 0-4o C for i0 rains. After centrifugation at 1000 g for 15 rains, aliquots of i00 ~i of the resulting supernatant were transferred to i0 ml of scintillation fluid. The radioactivity was counted in a scintillation spectrometer (SL40 Intertechnique). b) Total Binding Sites. In order to saturate all the DHT binding sites, cytosol stripped of free endogenous hormone was incubated with i0 nM DHT at 0-4 °C for 1 h. The excess of DHT was then removed by adding charcoal buffer (solution i) in a ratio of 1 : i0 v/v. Following centrifugation at i000 g for 20 rains aliquots of i00 ~i of the supernatant were removed for protein estimation (8). The remaining cytosol was then subjected to exchange assay as follows: Portions (100 ~i) of the saturated cytosol were incubated with (3H) R1881 (20-50 nM) in the presence or absence of 5000 nM radioinert R1881 at 15°C for 16 h. Removal of free from bound (3 H) R1881 was achieved by adding 50 ~i of dextran charcoal buffer (solution 2) at 0-4 ° C for i0 rains. After centrifugation at i000 g for 15 rains aliquots of I00 ~I of the supernatant were transferred to i0 ml scintillation fluid and the radioactivity counted in a scintillation spectrometer. Analysis
of the Results
The difference in counts/rain between cytosol labelled with (3H) R1881 and (3H) R1881 + R1881 was taken as a measure of the quantity of DHT receptor in the cytosol. It was, there-
R. G h a n a d i a n et al. : A n d r o g e n Receptors in the M o n k e y
Prostate
171
12Void volume
g
/L/
x
g ,34-
I
I
I
I
I
I
I
I
I0
20
30
~.0
50
60
70
80
i
90
100
Fraction No. (4 ml)
Fig. I. Sepharose 6B gel exclusion chromatography of the steroid receptor complex from the caudal zone of the monkey prostate
Table i. Effect of enzymic digestion complex. Analysis on Sephadex G-25
on receptor
220
Percentage reduction in binding
200-
................... ~g
180-
i
en
= 160-
Caudal
Cranial
DNase
* ND
ND
RNase
ND
ND
Pronase
g '5
Enzyme
~
140-
120-
I00~
D
e ,
x ,
~ t
r
bulin
a
,
n
%,Blue
I
2
Fig. 2. Standard - Sepharose 6B
3
4 5 -5 I0 M W xlO (daltons)
curve
i00 %
96 %
rritin
for molecular
20
weights
fore, possible to express the final results in t e r m s of f e m t o m o l e of specific receptor per m g of cytoplasmic protein. The difference between the total and unoccupied binding sites w a s considered as bound receptor (6).
RESULTS i. Characterisation of the Receptor The labelled cytosol prepared f r o m caudal and cranial lobes revealed two distinct peaks of
* Not detectable S a m p l e s (i00 ~i) of cytosol previously labelled with (3H) D H T as described w e r e incubated alone or together with 20 ~g of either pronase, deoxyribonuclease or ribonuclease at 37 ° C for 30 rain. T h e s a m p l e s w e r e then subjected to c h r o m a t o g r a p h y on Sephadex G - 2 5 c o l u m n (20 x 1.5 cm) eluted with buffer A at 2°C. Radioactivity associated with material eluted with the void v o l u m e of the c o l u m n w a s counted and c o m p a r e d to the control.
radioactivity (Fig. i). The first peak, which w a s elutedbetween fractions 40 and 50, w a s totally abolished by an excess of unlabelled dihydrotestosterone. T h e second peak, which corresponded to free steroid, w a s not affected. The effect of e n z y m e s on the elution profile s h o w e d that pronase completely abolished the first peak in the preparation f r o m each lobe but R N a s e and D N a s e did not affect this peak (Table i). T h e molecular weight of the steroid receptor c o m p l e x w a s estimated to be in the order of 2.8-2.9 x 10 5 daltons (Fig. 2).
172
Urol.
Table 2. Concentration prostate
Monkey (year)
of androgen
receptors
in the cytosol
Cranial lobe
Caudal lobe
fmol/mg protein
fmol/mg protein
total
free
bound
total
free
bound
Age
1
9
39.7
2.0
37.9
112.7
3.7
109.0
2
9
41.5
2.6
38.9
101.3
6.6
94.7
3
6
55.0
7.7
47.3
67.0
23.7
43.3
4
2
21.2
2.1
19.1
36.0
6.3
of Androgen
Receptors
The concentration for the total, free and bound receptors in 4 monkeys showed that the receptor content of the caudal zone was higher than in the cranial zone (Table 2). This difference was especially significant in the first two monkeys. The total receptor population in the third monkey was low but, comparatively, the free receptor was high. Although this monkey w a s a m a t u r e animal it w a s noted that the testes w e r e small. The fourth m o n k e y w a s i m m a t u r e and had a low level of receptors in both lobes. However, as with the m a t u r e monkey, the concentration of the receptor in the caudal lobe w a s higher than the cranial lobe. DISCUSSION These studies have demonstrated an androgen binding component in the caudal and cranial lobes of the monkey prostate. The inhibitory effect of unlabelled dihydrotestosterone on the first peak and not the second suggests that the first peak is an androgen binding component. This, however, requires further investigation using other steroids to demonstrate the binding specificity. The effect of enzymes on this steroid binding component suggests that the nature of the "receptor" is a protein. The results have shown that the behaviour of this receptor in the caudal and cranial lobes is similar. Furthermore, the molecular weight of this receptor is of the same order of magnitude as that of rat (9) and human prostate (unpublished results). Further studies are required to identify the labelled steroid bound to the receptor as well as the sedimentation constant of this-steroid receptor complex. The procedure for the measurement of the free and bound receptors for androgens in this study was
Vol.
5, No.
4 (1977)
of monkey
No
2. Concentration
Res.,
29.7
basically similar to that in our previous report (5) in which tritiated methyltrienolone, a synthetic androgen, w a s used in preference to labelled dihydrotestosterone. In the h u m a n prostate this c o m p o u n d specifically binds to dihydrotestosterone receptor protein and not to sex hormone binding globulin. Although in the present study the existence of SHBG in the monkey prostate has not been investigated, the fact that methyltrienolone unlike DHT does not undergo metabolism, makes this compound more advantageous for receptor m e a s u r e m e n t . It provides suitable conditions for an exchange assay which allow the m a x i m u m labelling of the receptor population. The specificity of R 1 8 8 1 for androgen receptors has been reported elsewhere (5). In earlier studies f r o m this laboratory it w a s demonstrated that the ability of the caudal lobe to take up and retain androgens w a s significantly higher than the cranial lobe (4). T h e present study supports our earlier findings and suggests that the caudal lobe contains m o r e free and bound androgen receptors. T h e s e data indicate that the caudal lobe in the m o n k e y can be equated with the peripheral zone of the h u m a n prostate. The data are also in keeping with the observation (I) that in development, the acinar structure of the peripheral zone is delayed until puberty c o m p a r e d with the central zone suggesting the greater dependence of the f o r m e r on an androgen stimulus.
REFERENCES i. Blacklock, N.J. : Surgical a n a t o m y of the prostate. In: Scientific Foundations of Urology, Eds. Williams, D.I., Chisholm, G.D., Vol. If, p. 113. H e i n e m a n n : L o n d o n 1976
R. Ghanadian et al. : Androgen Receptors in the M o n k e y Prostate B. Blacklock, N.J., Bouskill, K.: The zonal anatomy of the prostate in m a n and in the Rhesus monkey. Urological Research 5, 163 (1977) 3. Ghanadian, R.: Endocrine control of the prostate: M e c h a n i s m of action of androgens. In: Scientific Foundations of Urology, Eds. Williams, D.I., Chisholm, G.D., Vol. II, p. 138. Heinemann: London 1976 4. Ghanadian, R., Smith, C.B., Blacklock, N. J. : Differential androgen uptake by the lobes of the rhesus m o n k e y prostate. British Journal of Urology (in press) 5. Ghanadian, R., Auf, G., Chaloner, P.J., Chisholm, G.D.: The m e a s u r e m e n t of the free and bound cytoplasmic receptors for dihydrotestosterone in benign hypertrophied h u m a n prostate. Journal of Steroid Biochemistry (in press) 6. Katzenellenbogen, J.A., Johnson, H.J. Jr., Carlson, K.E.: Studies on the uterine, cytoplasmic estrogen binding protein. T h e r m a l stability and ligand dissociation
173
rate. An assay of empty and filled sites by exchange. Biochemistry 12, 4092 (1973) 7. Lehmann, F.G.: Molekulargewichtsbestimm u n g e n durch gelchromatographie an Sepharose-6B. Clinics Chimica Acta 28, 335 (1970)
8. Lowry, O . H . , Rosebrough, M . J . , F a r r , A.L., Randall, R.J.: Protein m e a s u r e m e n t with the folin-phenol reagent. Journal of Biological Chemistry 193, 265 (1951) 9. Mainwaring, W. I. P. : A soluble androgen receptor in the cytoplasm of rat ventral prostate. Journal of Endocrinology 45, 531 (1969)
R. Ghanadian, Ph. D. Prostate Research Laboratory Urological Unit Royal Postgraduate Medical School H a m m e r s m i t h Hospital London W I 2 0 H S U.K.
Urological R e s e a r c h 5, 1 6 9 - 1 7 3 (1977)
Androgen Receptors in the Prostate of the Rhesus Monkey R. Ghanadian, G. Auf, C.B. Smith, G.D. Chisholm, and N.J. Blacklock 1 P r o s t a t e R e s e a r c h L a b o r a t o r y , U r o l o g i c a l Unit, Royal P o s t g r a d u a t e Hospital, London, and 1Royal Naval Hospital, Haslar, U.K.
Received:
M e d ic a l School, H a r n r n e r s r n i t h
July 29, 1977
S u m m a r y . T h e p r e s e n c e of a n a n d r o g e n r e c e p t o r p r o t e i n i n the s u p e r n a t a n t p r e p a r a t i o n of the p r o s t a t e f r o m R h e s u s m o n k e y ( m a r a c a r n u l a t t a ) i s d e s c r i b e d . T h e m o l e c u l a r w e i g h t of t h i s r e c e p t o r p r o t e i n w a s f o u n d to be 2 . 8 - 2 . 9 x 1 0 5 d a l t o n s . T h e l e v e l s of f r e e a n d b o u n d a n d r o g e n r e c e p t o r s w e r e m e a s u r e d i n the c a u d a l a n d c r a n i a l l o b e s of the p r o s t a t e b y a n e x c h a n g e a s s a y u s i n g r n e t h y l t r i e n o l o n e (R1881). T h e c o n c e n t r a t i o n of the f r e e b i n d i n g s i t e s i n the c a u d a l l o b e r a n g e d b e t w e e n 3 . 7 - 2 3 . 7 f m o l / r n g p r o t e i n . T h e v a l u e i n the c r a n i a l l o b e w a s 2 . 0 - 7 . 7 f m o l / r n g p r o t e i n . T h e t o t a l b i n d i n g s i t e s i n the c a u d a l l o b e r a n g e d b e t w e e n 3 6 . 0 - 1 1 2 . 7 a n d i n the c r a n i a l b e t w e e n 2 1 . 2 - 5 5 . 0 f m o l / m g . T h e b o u n d r e c e p t o r r a n g e d between 43.3-109.0 and 19.1-47.3 fmol/rn~ protein for caudal and cranial lobes respectively. The l e v e l of b o t h b o u n d a n d f r e e r e c e p t o r s w a s f o u n d to b e s i g n i f i c a n t l y h i g h e r i n the c a u d a l l o b e . T h i s d a t a s u g g e s t s t h a t the two l o b e s of the p r o s t a t e i n the R h e s u s m o n k e y c a n b e e q u a t e d w i t h t h e two z o n e s of the h u m a n p r o s t a t e i n r e s p e c t of a n d r o g e n r e s p o n s i v e n e s s . Key words: Androgen receptors
- Rhesus monkey - Prostate
T h e e x i s t e n c e of a r e c e p t o r p r o t e i n w h i c h b i n d s s p e c i f i c a l l y to d i h y d r o t e s t o s t e r o n e h a s b e e n d e m o n s t r a t e d i n the p r o s t a t e of s e v e r a l s p e c i e s (3). In a p r e l i m i n a r y s t u d y f r o m t h i s l a b o r a t o r y it w a s d e m o n s t r a t e d t h a t the c a u d a l a n d c r a n i a l l o b e s of the p r o s t a t e i n the R h e s u s m o n k e y ( m a r a c a m u l a t t a ) w e r e a b l e to t a k e up a n d r e t a i n a n d r o g e n a n d t h a t the u p t a k e of t e s t o s t e r one b y the c a u d a l l o b e of the p r o s t a t e w a s significantly higher than any other target or n o n - t a r g e t t i s s u e (4). B o t h i n the r e l a t i o n s h i p to s u r r o u n d i n g s t r u c t u r e s a n d i n t h e i r d i f f e r e n t i al h i s t o l o g i c a l c h a r a c t e r i s t i c s the c r a n i a l lobe of the p r o s t a t e of the m o n k e y a p p e a r s to b e h o m o l o g o u s w i t h the c e n t r a l z o n e of the h u m a n a n d the c a u d a l l o b e w i t h the p e r i p h e r a l z o n e (2). T h e p r e s e n t s t u d y w a s c a r r i e d out to c h a r a c t e r i s e a n d r o g e n r e c e p t o r s i n the c y t o s o l of R h e s u s m o n k e y p r o s t a t e a n d to d e t e r m i n e the c o n c e n t r a t i o n of t h e s e r e c e p t o r s i n the cranial and caudal lobes.
M A T E t ~ I A L S AND M E T H O D S Monkeys Rhesus monkeys (maraca mulatta) aged between 2 a n d 10 y e a r s , w e r e u s e d i n t h i s s t u d y .
- Receptor assay.
Prostate and other tissues w e r e dissected out whilst the animals w e r e under general anaesthesia. S a m p l e s w e r e kept in containers on ice and transported to the laboratory. All the operative procedures w e r e p e r f o r m e d between ii and 15 h.
Steroids (i~, 2~, 3H) 5a-dihydrostestosterone (3H)DHT (specific activity 58 Ci/mrnol; Radiochemical Centre, Arnersham, U.K.). Unlabelled 5~dihydrotestosterone (DHT) (Sigma Chemicals). (6,7-3H) rnethyltrienolone (3H) R1881 (specific activity 55.5 Ci/rnmol) and unlabelled R1881 were kindly supplied by Roussel Uclaf, France.
Buffers
Buffer A: Tris buffer (20 rnM) containing 1.5 mM EDTA and 2 rnM rnereaptoethanol adjusted to pH 7.4. Buffer B: Tris buffer (i0 rnM) containing 1.5 rnM EDTA and 250 rnM sucrose adjusted to pH 7.4. Both buffers were freshly prepared and stored at 4°C
170
Urol.
Two solutions (i and 2) of dextran coated charcoal were prepared in buffer B. Solution 1 consisted of 5 % water washed Norit GSX charcoal (Hopkins and Williams) and 0.5% dextran T-70 (Pharmacia). Solution 2 consisted of 1.25% water washed Norit GSX charcoal and 0.625 % dextran T-70. Both solutions were stored at 4°C.
Solvents Ethanol, Methanol, Toluene and Triton X-100 were all of Analar grade (British D r u g Houses).
DNase and RNase from bovine pancreas and pronase from streptomyces griseus (Sigma Chemicals). Column
Chromatography
Sepharose
6B
(Pharmacia
Fine
Chemicals).
Scintillation Fluid Toluene: Triton X-100 (2 : i) containing 0.4% 2,5-diphenyloxaZole (Koch-Light Laboratories). Preparation
of Cytosol
Sections of either caudal or cranial lobes of the prostate were sliced, minced, blotted with filter paper and weighed. The minced tissue was placed in a pre-cooled B24 tube and homogenised in buffer (1:6 w/v). Homogenisationwas carried out using an Ultra Turrax type TPI8-10 (Junks and Kunkle. AG) using 5 strokes each of I0 seconds with 30 seconds cooling intervals. The homogenate was then centrifuged at 105,000 g for 1 h at 4 ° C. The resulting supernatant (cytosol fraction) was cleanly pipetted taking special care to avoid contamination from the lipid layer on the top. All the above procedures were carried out at 0-4 °C. Free endogenous hormone was removed from the cytosol fraction by incubating the cytosol with charcoal buffer (solution i) in a ratio of i0:i v/v at 0-4 °C for 10 rains. The steroid bound to charcoal was precipitated by centrifugation at 3700 g at 4 ° C for 20 rains. Aliquots of i00 ~i of the cytosol were then used for protein estimations (8). The remaining cytosol was used for the characterisation and the assay of both unoccupied and total binding sites.
Characterisation of Receptor Aliquots of the cytosol stripped of endogenous h o r m o n e were incubated with 50 n M of (3H) dihydrotestosterone in the presence and absence of unlabelled dihydrotestosterone at 15 ° C for 16 h. Unbound steroids were r e m o v e d by incubating the cytosol with dextran charcoal solution 2 (2:1 v/v) at 0-4 ° C for I0 rains. Fol-
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5, No.
4 (1977)
lowing centrifugation at 1000 g for 15 mins the supernatant was eluted on a precalibrated column (55 cmx 2.5 cm) of Sepharose 6B (7) at a flow rate of 40 ml/h with buffer A. Fractions (4 ml) were collected in an LKB 7000 fraction collector. Aliquots (2 ml) of each fraction were transferred to scintillation vials and scintillation fluid (15 ml) was added. The radioactivity was counted in a scintillation spectrometer. The results were expressed as counts per minute per fraction. Measurement
Enzymes
Res.,
of the Receptors
a) Free Binding Sites. Unoccupied binding sites (free receptors) were measured in the cytosol fraction as follows: For triplicate assay 6 test tubes were set up, each pair containing 0.i pmol (3H) R1881 with and without 500 prnol radioinert R1881 in ethanol. The solutions were evaporated to dryness and subsequently transferred to crushed ice. Aliquots of 0.1 ml cytosol were added to each tube and incubated at 0-4o C for 1 h. Unbound R1881 was removed by adding 50 ~i dextran charoal (solution 2) which was then vortexed and incubated at 0-4o C for i0 rains. After centrifugation at 1000 g for 15 rains, aliquots of i00 ~i of the resulting supernatant were transferred to i0 ml of scintillation fluid. The radioactivity was counted in a scintillation spectrometer (SL40 Intertechnique). b) Total Binding Sites. In order to saturate all the DHT binding sites, cytosol stripped of free endogenous hormone was incubated with i0 nM DHT at 0-4 °C for 1 h. The excess of DHT was then removed by adding charcoal buffer (solution i) in a ratio of 1 : i0 v/v. Following centrifugation at i000 g for 20 rains aliquots of i00 ~i of the supernatant were removed for protein estimation (8). The remaining cytosol was then subjected to exchange assay as follows: Portions (100 ~i) of the saturated cytosol were incubated with (3H) R1881 (20-50 nM) in the presence or absence of 5000 nM radioinert R1881 at 15°C for 16 h. Removal of free from bound (3 H) R1881 was achieved by adding 50 ~i of dextran charcoal buffer (solution 2) at 0-4 ° C for i0 rains. After centrifugation at i000 g for 15 rains aliquots of I00 ~I of the supernatant were transferred to i0 ml scintillation fluid and the radioactivity counted in a scintillation spectrometer. Analysis
of the Results
The difference in counts/rain between cytosol labelled with (3H) R1881 and (3H) R1881 + R1881 was taken as a measure of the quantity of DHT receptor in the cytosol. It was, there-
R. G h a n a d i a n et al. : A n d r o g e n Receptors in the M o n k e y
Prostate
171
12Void volume
g
/L/
x
g ,34-
I
I
I
I
I
I
I
I
I0
20
30
~.0
50
60
70
80
i
90
100
Fraction No. (4 ml)
Fig. I. Sepharose 6B gel exclusion chromatography of the steroid receptor complex from the caudal zone of the monkey prostate
Table i. Effect of enzymic digestion complex. Analysis on Sephadex G-25
on receptor
220
Percentage reduction in binding
200-
................... ~g
180-
i
en
= 160-
Caudal
Cranial
DNase
* ND
ND
RNase
ND
ND
Pronase
g '5
Enzyme
~
140-
120-
I00~
D
e ,
x ,
~ t
r
bulin
a
,
n
%,Blue
I
2
Fig. 2. Standard - Sepharose 6B
3
4 5 -5 I0 M W xlO (daltons)
curve
i00 %
96 %
rritin
for molecular
20
weights
fore, possible to express the final results in t e r m s of f e m t o m o l e of specific receptor per m g of cytoplasmic protein. The difference between the total and unoccupied binding sites w a s considered as bound receptor (6).
RESULTS i. Characterisation of the Receptor The labelled cytosol prepared f r o m caudal and cranial lobes revealed two distinct peaks of
* Not detectable S a m p l e s (i00 ~i) of cytosol previously labelled with (3H) D H T as described w e r e incubated alone or together with 20 ~g of either pronase, deoxyribonuclease or ribonuclease at 37 ° C for 30 rain. T h e s a m p l e s w e r e then subjected to c h r o m a t o g r a p h y on Sephadex G - 2 5 c o l u m n (20 x 1.5 cm) eluted with buffer A at 2°C. Radioactivity associated with material eluted with the void v o l u m e of the c o l u m n w a s counted and c o m p a r e d to the control.
radioactivity (Fig. i). The first peak, which w a s elutedbetween fractions 40 and 50, w a s totally abolished by an excess of unlabelled dihydrotestosterone. T h e second peak, which corresponded to free steroid, w a s not affected. The effect of e n z y m e s on the elution profile s h o w e d that pronase completely abolished the first peak in the preparation f r o m each lobe but R N a s e and D N a s e did not affect this peak (Table i). T h e molecular weight of the steroid receptor c o m p l e x w a s estimated to be in the order of 2.8-2.9 x 10 5 daltons (Fig. 2).
172
Urol.
Table 2. Concentration prostate
Monkey (year)
of androgen
receptors
in the cytosol
Cranial lobe
Caudal lobe
fmol/mg protein
fmol/mg protein
total
free
bound
total
free
bound
Age
1
9
39.7
2.0
37.9
112.7
3.7
109.0
2
9
41.5
2.6
38.9
101.3
6.6
94.7
3
6
55.0
7.7
47.3
67.0
23.7
43.3
4
2
21.2
2.1
19.1
36.0
6.3
of Androgen
Receptors
The concentration for the total, free and bound receptors in 4 monkeys showed that the receptor content of the caudal zone was higher than in the cranial zone (Table 2). This difference was especially significant in the first two monkeys. The total receptor population in the third monkey was low but, comparatively, the free receptor was high. Although this monkey w a s a m a t u r e animal it w a s noted that the testes w e r e small. The fourth m o n k e y w a s i m m a t u r e and had a low level of receptors in both lobes. However, as with the m a t u r e monkey, the concentration of the receptor in the caudal lobe w a s higher than the cranial lobe. DISCUSSION These studies have demonstrated an androgen binding component in the caudal and cranial lobes of the monkey prostate. The inhibitory effect of unlabelled dihydrotestosterone on the first peak and not the second suggests that the first peak is an androgen binding component. This, however, requires further investigation using other steroids to demonstrate the binding specificity. The effect of enzymes on this steroid binding component suggests that the nature of the "receptor" is a protein. The results have shown that the behaviour of this receptor in the caudal and cranial lobes is similar. Furthermore, the molecular weight of this receptor is of the same order of magnitude as that of rat (9) and human prostate (unpublished results). Further studies are required to identify the labelled steroid bound to the receptor as well as the sedimentation constant of this-steroid receptor complex. The procedure for the measurement of the free and bound receptors for androgens in this study was
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5, No.
4 (1977)
of monkey
No
2. Concentration
Res.,
29.7
basically similar to that in our previous report (5) in which tritiated methyltrienolone, a synthetic androgen, w a s used in preference to labelled dihydrotestosterone. In the h u m a n prostate this c o m p o u n d specifically binds to dihydrotestosterone receptor protein and not to sex hormone binding globulin. Although in the present study the existence of SHBG in the monkey prostate has not been investigated, the fact that methyltrienolone unlike DHT does not undergo metabolism, makes this compound more advantageous for receptor m e a s u r e m e n t . It provides suitable conditions for an exchange assay which allow the m a x i m u m labelling of the receptor population. The specificity of R 1 8 8 1 for androgen receptors has been reported elsewhere (5). In earlier studies f r o m this laboratory it w a s demonstrated that the ability of the caudal lobe to take up and retain androgens w a s significantly higher than the cranial lobe (4). T h e present study supports our earlier findings and suggests that the caudal lobe contains m o r e free and bound androgen receptors. T h e s e data indicate that the caudal lobe in the m o n k e y can be equated with the peripheral zone of the h u m a n prostate. The data are also in keeping with the observation (I) that in development, the acinar structure of the peripheral zone is delayed until puberty c o m p a r e d with the central zone suggesting the greater dependence of the f o r m e r on an androgen stimulus.
REFERENCES i. Blacklock, N.J. : Surgical a n a t o m y of the prostate. In: Scientific Foundations of Urology, Eds. Williams, D.I., Chisholm, G.D., Vol. If, p. 113. H e i n e m a n n : L o n d o n 1976
R. Ghanadian et al. : Androgen Receptors in the M o n k e y Prostate B. Blacklock, N.J., Bouskill, K.: The zonal anatomy of the prostate in m a n and in the Rhesus monkey. Urological Research 5, 163 (1977) 3. Ghanadian, R.: Endocrine control of the prostate: M e c h a n i s m of action of androgens. In: Scientific Foundations of Urology, Eds. Williams, D.I., Chisholm, G.D., Vol. II, p. 138. Heinemann: London 1976 4. Ghanadian, R., Smith, C.B., Blacklock, N. J. : Differential androgen uptake by the lobes of the rhesus m o n k e y prostate. British Journal of Urology (in press) 5. Ghanadian, R., Auf, G., Chaloner, P.J., Chisholm, G.D.: The m e a s u r e m e n t of the free and bound cytoplasmic receptors for dihydrotestosterone in benign hypertrophied h u m a n prostate. Journal of Steroid Biochemistry (in press) 6. Katzenellenbogen, J.A., Johnson, H.J. Jr., Carlson, K.E.: Studies on the uterine, cytoplasmic estrogen binding protein. T h e r m a l stability and ligand dissociation
173
rate. An assay of empty and filled sites by exchange. Biochemistry 12, 4092 (1973) 7. Lehmann, F.G.: Molekulargewichtsbestimm u n g e n durch gelchromatographie an Sepharose-6B. Clinics Chimica Acta 28, 335 (1970)
8. Lowry, O . H . , Rosebrough, M . J . , F a r r , A.L., Randall, R.J.: Protein m e a s u r e m e n t with the folin-phenol reagent. Journal of Biological Chemistry 193, 265 (1951) 9. Mainwaring, W. I. P. : A soluble androgen receptor in the cytoplasm of rat ventral prostate. Journal of Endocrinology 45, 531 (1969)
R. Ghanadian, Ph. D. Prostate Research Laboratory Urological Unit Royal Postgraduate Medical School H a m m e r s m i t h Hospital London W I 2 0 H S U.K.
