- Email: [email protected]
Subcellular location of androgen receptor in rat prostate, seminal vesicle and human osteosarcoma MG-63 cells
J. SteroidBiochem. Molec. BioLVol.41, No. 3-8, pp. 693~96, 1992
0960-0760/92 $5.00+ 0.00 Copyright© 1992PergamonPressplc
Printed in Great Britain.All rights reserved
SUBCELLULAR IN
RAT
LOCATION
PROSTATE,
OF
SEMINAL
ANDROGEN VESICLE
OSTEOSARCOMA
MG-63
RECEPTOR AND
HUMAN
CELLS
Y. H. ZHUANG,M. BL,~,UER,A. PEKKIand P. TUOHIMAA* Department of Biomedical Sciences, University of Tampere, 33101 Tampere, Finland Summary--Location of the androgen receptor (AR) before and after dihydrotestosterone (DHT) administration was studied in 6 castrated and 2 normal male rats, as well as in MG-63 human osteosarcoma cell culture. Two days after castration, rats were injected with DHT and sacrificed 0, 6 and 24 h later. Cryosections of ventral prostate and seminal vesicle were stained with a polyclonal anti-AR antibody. Cultured MG-63 cells were also stained similarly. The intensity of immunoreaction was measured semiquantitatively by computer-assisted image analysis. In both normal and castrated rats, a positive reaction was seen mainly in the nuclei of epithelial cells and stromal cells of the prostate and seminal vesicle, as well as in those of smooth muscle cells of the seminal vesicle. AR immunoreactivity was up-regulated by DHT, it decreased clearly in both organs after castration. Nuclear AR and its up-regulation by androgen were also seen in MG-63 cells. At the immunoelectron microscopy, silver enhanced gold particles were predominantly found in the heterochromatin of cell nuclei. Treatment with DHT caused a decondensation of the heterochromatin and AR was more dispersed. Thus, AR appears to be nuclear independently of the ligand.
INTRODUCTION
EXPERIMENTAL
Androgens, through a specific receptor (AR), play an important role in the development and maintenance of the function of many target organs, especially in the male sex and accessory sex organs. Recent clonings of human and rat A R c D N A s have revealed the amino acid sequences for respective ARs, which has allowed the development of specific antibodies against AR[1]. Although A R is regulated by many steroids, there are controversial results about androgen effects on its own receptor. A recent study suggests that m R N A of A R and the amount of the A R protein increase after castration and decrease after treatment with androgen in the rat prostate and seminal vesicle [2], while immunohistochemical studies show opposite results in both organs [3]. In this study, we examined A R as well as its regulation by dihydrotestosterone ( D H T ) in the prostate and seminal vesicle of both intact and castrated rats, and a MG-63 osteosarcoma cell line with light and electron microscopic immunohistochemical methods.
Animals and tissues
Eight Sprague-Dawley rats at 3 months of age were used. Six of them were castrated under ether anaesthesia and the other two remained as normal controls. The castrated rats were injected with D H T (100 tag/per rat, dissolved in 20% ethanol in PBS) s.c. 48 h after castration. Rats were sacrificed by decapitation 0, 6 and 24 h after treatment with D H T . The ventral prostates and seminal vesicles of both intact and castrated rats were taken for light and electron microscopy. Cell culture
A human osteosarcoma cell line, MG-63 (ATCC, Rockville, MA, U.S.A.), was cultured for 1 day[4] after which D H T (1 taM) was added to the cell culture medium for 24, 6, 2 and 0.5 h. Cells were fixed in Baker's fixative (4% formaldehyde, 1% CaC12"2H20 p H 6.7) for 15min and permeabilized with 0.5% Triton X-100 for 40 min.
Proceedings of the lOth International Symposium of the Journal of Steroid Biochemistry and Molecular Biology, Recent Advances in Steroid Biochemistry and Molecular Biology, Paris, France, 26-29 May 1991.
*To whom correspondence should be addressed.
Immunohistochemistry and immunoelectron microscopy
The polyclonal anti-AR antibody, produced against a sequence of 242 amino acids of the human A R starting from the amino acid num-
693
694
Y.H. ZI'IUANGet al.
ber 331 [5], was kindly provided by Dr S. Liao, University of Chicago. The working concentration of the antibody was 2 #g/ml for both light and electron microscopy. Frozen sections (10#m) of ventral prostate and seminal vesicle were fixed in 3.7% formaldehyde for 15 min followed by PBS for 5 min, absolute methanol and acetone for 4 and 2 min at -20°C, respectively. Sections were stained using the avidin-biotin method as described previously [6]. For controls, the primary antibody was preabsorbed with rat ventral prostate cytosol for 4 h at 4°C or replaced by PBS. Small pieces of prostate and seminal vesicle were fixed in Faglu fixative (4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M PBS pH 7.4) for 2h. 100#m sections were cut in a vibratom. The pre-embedding modification of immunogold-silver method and processing for electron microscopy were carried out as described in detail elsewhere [7]. After incubation with primary and secondary antibodies, the sections were incubated for 50 min with 5 nm gold particles (Janssen Pharmaceuticals, Beerse, Belgium) coupled to streptavidin, followed by silver intensification solution (Janssen Life Sciences Products, Olen, Belgium) for 10 min. U1trathin sections were examined with JEOL 1200 EX electron microscope.
in the epithelial than in the stromal cells, while in the seminal vesicle it was more intense in smooth muscle than in epithelial cells. In the castrated rats, the reaction showed the same pattern as in intact ones, but it decreased clearly in both the prostate and seminal vesicle of rats after castration and without DHT treatment (0 h), increased in all cell types in both organs 6 h after injection of hormone. With image analysis, there was a statistically significant difference (P < 0.05) between intact controis and castrated rats. Figure 2(A) shows that prostatic epithelial cells responded more than the stromal cells to castration and hormone treatment. At 24 h after DHT, the two organs showed different response, AR expression remained high in the prostate, whereas in the seminal vesicle it decreased. At the immunoelectron microscopic level, most silver enhanced gold particles were detected in cell nuclei in both ventral prostate and seminal vesicle. The most intense labeling was found in the heterochromatin [Fig. I(C)]. In castrated rats, 6 h after treatment with DHT, the heterochromatin became less condensed [P < 0.05, Fig. 2(B)] and more silver enhanced gold particles were seen in the euchromatin than in non-treated rats.
Image analysis
With light microscopy, the MG-63 osteosarcoma cells also showed nuclear staining at both hormone-treated and untreated cells; cytoplasmic reaction was hardly seen [Fig. I(D)]. The reaction was slightly weaker 0.5 h after treatment with DHT than without hormone. At 2 h staining increased and remained high at 6 and 24 h.
In order to quantify the immunostaining, the stained cells were analyzed using a computerassisted image analysis system (Java Dialog Program, Jandel Scientific, CA, U.S.A.) as described previously [8]. The same method was used to measure the amount of heterochromatin in prostatic epithelial cells of DHT-treated and non-treated castrated rat. Mean and standard deviation of each group were calculated and compared by Student's t-test. RESULTS Prostate and seminal vesicle
With light microscopy, in both ventral prostate and seminal vesicle of intact rats, the immunopositive staining was located mainly in the nuclei of epithelial, stromal as well as smooth muscle cells [Fig. I(A)] Some cytoplasmic staining was seen, but it was at least partially due to non-specific staining. Control sections showed no nuclear staining [Fig. I(B)]. In the prostate the reaction was more intense
M G - 6 3 osteosarcoma cell line
DISCUSSION In this study we demonstrated that AR is located mainly in the cellular nuclei of the ventral prostate and seminal vesicle in both intact and castrated rats in both light and electron microscopic level. The result is in agreement with previous reports of autoradiographic and immunohistochemical studies [3, 9]. Although practically all cell types in both organs contained AR, the distribution and intensity varied. Epithelial nuclear AR staining was strong in the ventral prostate, but moderate in the seminal vesicle. In the seminal vesicle the immunostaining of smooth muscle cell nuclei was stronger than in epithelial cells, which has
Subcellular location of androgen receptor in rat and human cells
695
Fig. 1. Location of AR in the rat prostate: (A) the immunopositivestaining is mainlylocated in the nuclei of epithelial cells in the intact rat. (arrowhead) Bar 20 #m. (B) Control staining with primary antibody preabsorbed with prostatic cytosol. No positive reaction is seen. Bar 20/~m. (C) Immunoeleetron microscopy of castrated rat prostate epithelial cell 6 h after treatment with DHT, silver enhanced gold particles are mainly located in the heterochromatin.Arrowheads: nuclear membrane. Bar 0.2 #m. (D) Immunostaining of AR in human MG-63 osteosarcomacells 2 h after treatment with DHT. Bar 20 #m. also been observed by other authors [9]. Varying expression of AR in different types of cells suggests that the cells have different AR contents and different responses to androgen. Our results demonstrated that AR is downregulated within 2days after castration and up-regulated by DHT. We obtained similar results in rat accessory sex glands and human osteosarcoma cells. In agreement with this study, Sar et al. [3] found that 3 days after castration the rat ventral prostate, seminal vesicle, epididymis and ductus deferens lost their A R immunostaining, which was restored within 15 min of androgen administration. This upregulation by homologous ligand is similar to the vitamin D receptor (VDR), but different from the estrogen, glucocorticoid and progesterone receptors which are down-regulated by occupancy of the receptor [10-14]. The mechanism of AR down-regulation after withdrawal of androgen and its up-regulation by hormone treatment is still unknown. It is possible that the epitope studied here shows conformational changes, so that it may or may not be recog-
nized by the antibody. It has been shown that the m R N A of AR as well as the AR protein concentration in rat ventral prostate and seminal vesicle increases after castration and decreases 24-48 h after administration of androgen [2]. Thus, AR seems to be regulated by androgen. However, different cells seem to have a different response to stimulation with androgen; in Sertoli cells testosterone increased in A R protein concentration 2-3 fold, but did not increase the expression of the AR m R N A [15]. This is the first report showing the location of AR at the immunoelectron microscopic level. With the same method, Isola [7] previously described immunoelectron microscopic localization of progesterone receptor (PR) in the chick oviduct. The location of AR predominantly in the heterochromatin is similar to that of PR and the heterochromatin was shown to be dispersed after treatment with progesterone in the chick and D H T in castrated rats. Treatment with D H T induced the expression of AR and changed its location, so that more labeling appeared in the euchromatin after treatment
696
Y.H. ZHUANG et al. from the Sigrid Juselius Foundation and the Academy of Finland.
(A) Prostote Epit heliol
100
J77] Mesenchyrnol
I
80
cell cell
60
~
40
20
//
/
// // // // //
/ / / / /
// //
/ /
/ -/ N
•1 O
REFERENCES
// / / // / /
// // // / /
/A 24
//
Time a f t e r DHT (h}
60-
(B) Prostate
50
m
40 o
T
o
3O
20
10 Oh
6h
Fig. 2. Quantification by computer assisted-image analysis of AR and heterochromatin in rat prostate: (A) immunoreaction is reduced after castration and increased after treatment with DHT. N = non-treated normal control. 0, 6 and 24 h equal the time after treatment with DHT in castrated rats. (B) Amount of heterochromatin in prostate epithelial cells is reduced after treatment with DHT in castrated rats.
with DHT than without DHT. It may be because androgen activates the transcription of specific genes, and localization change of AR could be a result of conformational changes in the AR-bound chromatin. Acknowledgements--The authors thank Dr Shutsung Liao from the University of Chicago for kindly providing the antibody, Ms Lenarita Peltonen, Mrs Tarja Arvela for skilful technical assistance, and Mr Robert McGilleon for revising the language. The work was supported by grants
I. Chang C., Kokontis J. and Liao S.: Molecular cloning of human and rat complementary DNA encoding androgen receptor. Science 240 (1989) 324-326. 2. Shan L. X., Rodriguez C. and J/inne O. A.: Regulation of androgen receptor protein and mRNA concentration by androgens in rat ventral prostate and seminal vessicle and in human hepatoma cells. Molec Endocr. 4 (1990) 1636-1646. 3. Sar M., Lubahn D. B., French F. S. and Wilson E. M.: Immunohistochemical localization of the androgen receptor in rat and human tissues. Endocrinology 127 (1990) 3180-3186. 4. Mahonen A., Pirskanen A., Kein~inen and M/ienp/i/i P. H.: Effect of 1, 25 (OH)2 D3 on its receptor mRNA levels and osteocalcin synthesis in human osteosarcoma cells. Biochim. Biophys. Acta 1048 (1990) 30-37. 5. Chang C., Whelan C. T., Popovich T. C. and Kokontis J.: Fusion proteins containing androgen receptor sequences and their use in the production of poly- and monoclonal anti-androgen receptor. Endocrinology 123 (1989) 1097-1099. 6. Ylikomi T., Gasc J. M., Isola J., Baulieu E. E. and Tuohimaa P.: Progesterone receptor in the chick bursa of Fabricius: characterization and immunohistochemical localization. Endocrinology 117 (1985) 155-160. 7. Isola, J. J.: The effect of progesterone on the localization of progesterone receptors in the nuclei of chick oviduct cells. Cell Tissue Res. 249 (1987) 317-323. 8. Pekki A., Bl/iuer M., Syv~il/i H., M/ikinen E.-K., Ylikomi T. and Tuohimaa P.: Immunological analysis of down-regulation of the chicken progesterone receptor by ligand. Submitted. 9. Bl~iuer M., Vaalasti A., Pauli S. L., Ylikomi T., Joensuu T. and Tuohimaa P.: Location of androgen receptor in human skin. J. Invest. Dermat. (1991). In press. 10. Smith R. G., Syms A. J., Nag A., Lerner S. and Norris S.: Mechanism of the glucocorticoid regulation of growth of the androgen-sensitive prostate-derived R3327-A1 tumor cell line. J. Biol. Chem. 260 (1985) 12454-I 2463. 1I. Costa E. M., Hirst M. A. and Feldman D.: Regulation of 1, 25-dihydroxyvitamin D3 receptors by vitamin D analogs in cultured mammalian cells. Endocrinology 117 (1985) 2203-2210. 12. Saceda M., Lippman M. E., Chambon P. and Lindsey R. L.: Regulation of the estrogen receptor in MCF-7 cells by estradiol. Molec. Endocr. 2 (1988) 1157-1162. 13. Rosewicz S., McDonald A. R., Maddux B. A., Goldfine I. D., Miesfeld R. L. and Logsdon C. D.: Mechanism of glucocorticoid receptor down-regulation by glucocorticoids. J. Biol. Chem. (1988) 2581-2584. 14. Tuohimaa P., Joensuu T., lsola J., Kein~.nen R., Kunnas T., Niemel/i A., Pekki A., Wallen M., Ylikomi M. and Kulomaa M.: Development of progestin-specific response in the chicken oviduct. Int. J. Def. Biol. 33 (1989) 125-134. 15. Blok L. J., Mackenbach P., Trapman J., Themmen A. P., Brinkmann A. O. and Grootegoed J. A.: Folliclestimulating hormone regulates androgen receptor mRNA in Sertoli cells. Molec. Cell Endocr. (1989) 267-271.
0960-0760/92 $5.00+ 0.00 Copyright© 1992PergamonPressplc
Printed in Great Britain.All rights reserved
SUBCELLULAR IN
RAT
LOCATION
PROSTATE,
OF
SEMINAL
ANDROGEN VESICLE
OSTEOSARCOMA
MG-63
RECEPTOR AND
HUMAN
CELLS
Y. H. ZHUANG,M. BL,~,UER,A. PEKKIand P. TUOHIMAA* Department of Biomedical Sciences, University of Tampere, 33101 Tampere, Finland Summary--Location of the androgen receptor (AR) before and after dihydrotestosterone (DHT) administration was studied in 6 castrated and 2 normal male rats, as well as in MG-63 human osteosarcoma cell culture. Two days after castration, rats were injected with DHT and sacrificed 0, 6 and 24 h later. Cryosections of ventral prostate and seminal vesicle were stained with a polyclonal anti-AR antibody. Cultured MG-63 cells were also stained similarly. The intensity of immunoreaction was measured semiquantitatively by computer-assisted image analysis. In both normal and castrated rats, a positive reaction was seen mainly in the nuclei of epithelial cells and stromal cells of the prostate and seminal vesicle, as well as in those of smooth muscle cells of the seminal vesicle. AR immunoreactivity was up-regulated by DHT, it decreased clearly in both organs after castration. Nuclear AR and its up-regulation by androgen were also seen in MG-63 cells. At the immunoelectron microscopy, silver enhanced gold particles were predominantly found in the heterochromatin of cell nuclei. Treatment with DHT caused a decondensation of the heterochromatin and AR was more dispersed. Thus, AR appears to be nuclear independently of the ligand.
INTRODUCTION
EXPERIMENTAL
Androgens, through a specific receptor (AR), play an important role in the development and maintenance of the function of many target organs, especially in the male sex and accessory sex organs. Recent clonings of human and rat A R c D N A s have revealed the amino acid sequences for respective ARs, which has allowed the development of specific antibodies against AR[1]. Although A R is regulated by many steroids, there are controversial results about androgen effects on its own receptor. A recent study suggests that m R N A of A R and the amount of the A R protein increase after castration and decrease after treatment with androgen in the rat prostate and seminal vesicle [2], while immunohistochemical studies show opposite results in both organs [3]. In this study, we examined A R as well as its regulation by dihydrotestosterone ( D H T ) in the prostate and seminal vesicle of both intact and castrated rats, and a MG-63 osteosarcoma cell line with light and electron microscopic immunohistochemical methods.
Animals and tissues
Eight Sprague-Dawley rats at 3 months of age were used. Six of them were castrated under ether anaesthesia and the other two remained as normal controls. The castrated rats were injected with D H T (100 tag/per rat, dissolved in 20% ethanol in PBS) s.c. 48 h after castration. Rats were sacrificed by decapitation 0, 6 and 24 h after treatment with D H T . The ventral prostates and seminal vesicles of both intact and castrated rats were taken for light and electron microscopy. Cell culture
A human osteosarcoma cell line, MG-63 (ATCC, Rockville, MA, U.S.A.), was cultured for 1 day[4] after which D H T (1 taM) was added to the cell culture medium for 24, 6, 2 and 0.5 h. Cells were fixed in Baker's fixative (4% formaldehyde, 1% CaC12"2H20 p H 6.7) for 15min and permeabilized with 0.5% Triton X-100 for 40 min.
Proceedings of the lOth International Symposium of the Journal of Steroid Biochemistry and Molecular Biology, Recent Advances in Steroid Biochemistry and Molecular Biology, Paris, France, 26-29 May 1991.
*To whom correspondence should be addressed.
Immunohistochemistry and immunoelectron microscopy
The polyclonal anti-AR antibody, produced against a sequence of 242 amino acids of the human A R starting from the amino acid num-
693
694
Y.H. ZI'IUANGet al.
ber 331 [5], was kindly provided by Dr S. Liao, University of Chicago. The working concentration of the antibody was 2 #g/ml for both light and electron microscopy. Frozen sections (10#m) of ventral prostate and seminal vesicle were fixed in 3.7% formaldehyde for 15 min followed by PBS for 5 min, absolute methanol and acetone for 4 and 2 min at -20°C, respectively. Sections were stained using the avidin-biotin method as described previously [6]. For controls, the primary antibody was preabsorbed with rat ventral prostate cytosol for 4 h at 4°C or replaced by PBS. Small pieces of prostate and seminal vesicle were fixed in Faglu fixative (4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M PBS pH 7.4) for 2h. 100#m sections were cut in a vibratom. The pre-embedding modification of immunogold-silver method and processing for electron microscopy were carried out as described in detail elsewhere [7]. After incubation with primary and secondary antibodies, the sections were incubated for 50 min with 5 nm gold particles (Janssen Pharmaceuticals, Beerse, Belgium) coupled to streptavidin, followed by silver intensification solution (Janssen Life Sciences Products, Olen, Belgium) for 10 min. U1trathin sections were examined with JEOL 1200 EX electron microscope.
in the epithelial than in the stromal cells, while in the seminal vesicle it was more intense in smooth muscle than in epithelial cells. In the castrated rats, the reaction showed the same pattern as in intact ones, but it decreased clearly in both the prostate and seminal vesicle of rats after castration and without DHT treatment (0 h), increased in all cell types in both organs 6 h after injection of hormone. With image analysis, there was a statistically significant difference (P < 0.05) between intact controis and castrated rats. Figure 2(A) shows that prostatic epithelial cells responded more than the stromal cells to castration and hormone treatment. At 24 h after DHT, the two organs showed different response, AR expression remained high in the prostate, whereas in the seminal vesicle it decreased. At the immunoelectron microscopic level, most silver enhanced gold particles were detected in cell nuclei in both ventral prostate and seminal vesicle. The most intense labeling was found in the heterochromatin [Fig. I(C)]. In castrated rats, 6 h after treatment with DHT, the heterochromatin became less condensed [P < 0.05, Fig. 2(B)] and more silver enhanced gold particles were seen in the euchromatin than in non-treated rats.
Image analysis
With light microscopy, the MG-63 osteosarcoma cells also showed nuclear staining at both hormone-treated and untreated cells; cytoplasmic reaction was hardly seen [Fig. I(D)]. The reaction was slightly weaker 0.5 h after treatment with DHT than without hormone. At 2 h staining increased and remained high at 6 and 24 h.
In order to quantify the immunostaining, the stained cells were analyzed using a computerassisted image analysis system (Java Dialog Program, Jandel Scientific, CA, U.S.A.) as described previously [8]. The same method was used to measure the amount of heterochromatin in prostatic epithelial cells of DHT-treated and non-treated castrated rat. Mean and standard deviation of each group were calculated and compared by Student's t-test. RESULTS Prostate and seminal vesicle
With light microscopy, in both ventral prostate and seminal vesicle of intact rats, the immunopositive staining was located mainly in the nuclei of epithelial, stromal as well as smooth muscle cells [Fig. I(A)] Some cytoplasmic staining was seen, but it was at least partially due to non-specific staining. Control sections showed no nuclear staining [Fig. I(B)]. In the prostate the reaction was more intense
M G - 6 3 osteosarcoma cell line
DISCUSSION In this study we demonstrated that AR is located mainly in the cellular nuclei of the ventral prostate and seminal vesicle in both intact and castrated rats in both light and electron microscopic level. The result is in agreement with previous reports of autoradiographic and immunohistochemical studies [3, 9]. Although practically all cell types in both organs contained AR, the distribution and intensity varied. Epithelial nuclear AR staining was strong in the ventral prostate, but moderate in the seminal vesicle. In the seminal vesicle the immunostaining of smooth muscle cell nuclei was stronger than in epithelial cells, which has
Subcellular location of androgen receptor in rat and human cells
695
Fig. 1. Location of AR in the rat prostate: (A) the immunopositivestaining is mainlylocated in the nuclei of epithelial cells in the intact rat. (arrowhead) Bar 20 #m. (B) Control staining with primary antibody preabsorbed with prostatic cytosol. No positive reaction is seen. Bar 20/~m. (C) Immunoeleetron microscopy of castrated rat prostate epithelial cell 6 h after treatment with DHT, silver enhanced gold particles are mainly located in the heterochromatin.Arrowheads: nuclear membrane. Bar 0.2 #m. (D) Immunostaining of AR in human MG-63 osteosarcomacells 2 h after treatment with DHT. Bar 20 #m. also been observed by other authors [9]. Varying expression of AR in different types of cells suggests that the cells have different AR contents and different responses to androgen. Our results demonstrated that AR is downregulated within 2days after castration and up-regulated by DHT. We obtained similar results in rat accessory sex glands and human osteosarcoma cells. In agreement with this study, Sar et al. [3] found that 3 days after castration the rat ventral prostate, seminal vesicle, epididymis and ductus deferens lost their A R immunostaining, which was restored within 15 min of androgen administration. This upregulation by homologous ligand is similar to the vitamin D receptor (VDR), but different from the estrogen, glucocorticoid and progesterone receptors which are down-regulated by occupancy of the receptor [10-14]. The mechanism of AR down-regulation after withdrawal of androgen and its up-regulation by hormone treatment is still unknown. It is possible that the epitope studied here shows conformational changes, so that it may or may not be recog-
nized by the antibody. It has been shown that the m R N A of AR as well as the AR protein concentration in rat ventral prostate and seminal vesicle increases after castration and decreases 24-48 h after administration of androgen [2]. Thus, AR seems to be regulated by androgen. However, different cells seem to have a different response to stimulation with androgen; in Sertoli cells testosterone increased in A R protein concentration 2-3 fold, but did not increase the expression of the AR m R N A [15]. This is the first report showing the location of AR at the immunoelectron microscopic level. With the same method, Isola [7] previously described immunoelectron microscopic localization of progesterone receptor (PR) in the chick oviduct. The location of AR predominantly in the heterochromatin is similar to that of PR and the heterochromatin was shown to be dispersed after treatment with progesterone in the chick and D H T in castrated rats. Treatment with D H T induced the expression of AR and changed its location, so that more labeling appeared in the euchromatin after treatment
696
Y.H. ZHUANG et al. from the Sigrid Juselius Foundation and the Academy of Finland.
(A) Prostote Epit heliol
100
J77] Mesenchyrnol
I
80
cell cell
60
~
40
20
//
/
// // // // //
/ / / / /
// //
/ /
/ -/ N
•1 O
REFERENCES
// / / // / /
// // // / /
/A 24
//
Time a f t e r DHT (h}
60-
(B) Prostate
50
m
40 o
T
o
3O
20
10 Oh
6h
Fig. 2. Quantification by computer assisted-image analysis of AR and heterochromatin in rat prostate: (A) immunoreaction is reduced after castration and increased after treatment with DHT. N = non-treated normal control. 0, 6 and 24 h equal the time after treatment with DHT in castrated rats. (B) Amount of heterochromatin in prostate epithelial cells is reduced after treatment with DHT in castrated rats.
with DHT than without DHT. It may be because androgen activates the transcription of specific genes, and localization change of AR could be a result of conformational changes in the AR-bound chromatin. Acknowledgements--The authors thank Dr Shutsung Liao from the University of Chicago for kindly providing the antibody, Ms Lenarita Peltonen, Mrs Tarja Arvela for skilful technical assistance, and Mr Robert McGilleon for revising the language. The work was supported by grants
I. Chang C., Kokontis J. and Liao S.: Molecular cloning of human and rat complementary DNA encoding androgen receptor. Science 240 (1989) 324-326. 2. Shan L. X., Rodriguez C. and J/inne O. A.: Regulation of androgen receptor protein and mRNA concentration by androgens in rat ventral prostate and seminal vessicle and in human hepatoma cells. Molec Endocr. 4 (1990) 1636-1646. 3. Sar M., Lubahn D. B., French F. S. and Wilson E. M.: Immunohistochemical localization of the androgen receptor in rat and human tissues. Endocrinology 127 (1990) 3180-3186. 4. Mahonen A., Pirskanen A., Kein~inen and M/ienp/i/i P. H.: Effect of 1, 25 (OH)2 D3 on its receptor mRNA levels and osteocalcin synthesis in human osteosarcoma cells. Biochim. Biophys. Acta 1048 (1990) 30-37. 5. Chang C., Whelan C. T., Popovich T. C. and Kokontis J.: Fusion proteins containing androgen receptor sequences and their use in the production of poly- and monoclonal anti-androgen receptor. Endocrinology 123 (1989) 1097-1099. 6. Ylikomi T., Gasc J. M., Isola J., Baulieu E. E. and Tuohimaa P.: Progesterone receptor in the chick bursa of Fabricius: characterization and immunohistochemical localization. Endocrinology 117 (1985) 155-160. 7. Isola, J. J.: The effect of progesterone on the localization of progesterone receptors in the nuclei of chick oviduct cells. Cell Tissue Res. 249 (1987) 317-323. 8. Pekki A., Bl/iuer M., Syv~il/i H., M/ikinen E.-K., Ylikomi T. and Tuohimaa P.: Immunological analysis of down-regulation of the chicken progesterone receptor by ligand. Submitted. 9. Bl~iuer M., Vaalasti A., Pauli S. L., Ylikomi T., Joensuu T. and Tuohimaa P.: Location of androgen receptor in human skin. J. Invest. Dermat. (1991). In press. 10. Smith R. G., Syms A. J., Nag A., Lerner S. and Norris S.: Mechanism of the glucocorticoid regulation of growth of the androgen-sensitive prostate-derived R3327-A1 tumor cell line. J. Biol. Chem. 260 (1985) 12454-I 2463. 1I. Costa E. M., Hirst M. A. and Feldman D.: Regulation of 1, 25-dihydroxyvitamin D3 receptors by vitamin D analogs in cultured mammalian cells. Endocrinology 117 (1985) 2203-2210. 12. Saceda M., Lippman M. E., Chambon P. and Lindsey R. L.: Regulation of the estrogen receptor in MCF-7 cells by estradiol. Molec. Endocr. 2 (1988) 1157-1162. 13. Rosewicz S., McDonald A. R., Maddux B. A., Goldfine I. D., Miesfeld R. L. and Logsdon C. D.: Mechanism of glucocorticoid receptor down-regulation by glucocorticoids. J. Biol. Chem. (1988) 2581-2584. 14. Tuohimaa P., Joensuu T., lsola J., Kein~.nen R., Kunnas T., Niemel/i A., Pekki A., Wallen M., Ylikomi M. and Kulomaa M.: Development of progestin-specific response in the chicken oviduct. Int. J. Def. Biol. 33 (1989) 125-134. 15. Blok L. J., Mackenbach P., Trapman J., Themmen A. P., Brinkmann A. O. and Grootegoed J. A.: Folliclestimulating hormone regulates androgen receptor mRNA in Sertoli cells. Molec. Cell Endocr. (1989) 267-271.