0013-7227/91/1284-1902$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 128, No. 4 Printed in U.S.A.
Androgen Receptor Expression in the Developing Rat Prostate Is Not Altered by Castration, Flutamide, or Suppression of the Adrenal Axis* DOUGLAS A. HUSMANNf, MICHAEL J. McPHAUL$, AND JEAN D. WILSON Departments of Surgery (Division of Urology) and Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9031
ABSTRACT. Mesenchymal epithelial interactions are believed to be important to the growth and development of the neonatal prostate. Prior studies in the rat ventral prostate, using autoradiography and tritiated dihydrotestosterone, indicate that androgen receptors are present in the prostatic stroma on day 3 and are detected in the epithelium by the tenth postnatal day. These findings suggested that androgen stimulation of the prostatic mesenchyme is a crucial step in the growth and development of the prostate. We have examined this developmental program directly using polyclonal antibodies that recognize specific epitopes of the androgen receptor to examine the pattern of androgen receptor expression in intact and neonatally castrate
M
ORPHOGENESIS of the rat ventral prostate is mediated by androgens, as shown by studies in which androgen ablation (1-3) before sexual differentiation prevents prostate development. In the neonatal period, prostatic growth is also androgen dependent, and castration of neonatal mice or rats inhibits the continued growth and development of the organ (4, 5). Androgens, however, do not appear to be the only factors controlling the growth of the prostate, since neonatal castration (4) or administration of cyproterone acetate (5, 6) does not prevent growth completely. The continued, albeit blunted, development of the prostate after neonatal androgen ablation could have several explanations. First, processes programmed by androgens during embryonic development may continue to operate postnatally in the absence of testicular androgen. Second, adrenal androgens acting upon the androgen receptors (7) may account for the apparent androgen-independent component of Received November 15,1990. Address all correspondence and requests for reprints to: Dr. D. A. Husmann, Division of Urology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 752359031. * This work was supported by grants from the NIH (DK-03892), the Perot Family Foundation, the March of Dimes (Basil O'Connor Award Grant 5-694), and the American Cancer Society (IN-142). t Scholar of the American Urologic Association. X Culpeper Medical Scholar.
animals. In keeping with previous studies, androgen receptors are present in the prostate stroma at birth and subsequently appear in the prostatic epithelium by the 10th postnatal day. Development of androgen receptor expression in the epithelium was not changed when the animals were castrated at birth, castrated and blocked by flutamide, or castrated and given hydrocortisone to suppress the production of adrenal androgens. These findings suggest that the appearance of androgen receptors in the prostatic epithelium is programmed by androgens before birth or that factors other than testicular or adrenal androgens control the development of epithelial androgen receptors. {Endocrinology 128: 1902-1906, 1991)
prostate growth. Finally, other hormones, such as GH, may play an independent role in the growth of the organ. As the action of androgens is mediated via specific intracellular receptors, considerable attention has been focused on the amount and distribution of the androgen receptor in the urogenital sinus and developing prostate. Studies of the stromal-epithelial interactive androgen receptor in the developing prostate have been based on autoradiography using tritiated dihydrotestosterone (713). These studies have demonstrated that the receptor is mesenchymal in location in the prenatal and early postnatal mouse and rat and is detectable within the prostate epithelium at the end of the first postpartum week when the prostatic ducts canalize (8, 9). This functional differentiation in which the receptor develops first in mesenchyme and then in the epithelium is thought to be androgen dependent (9). In the current work we have examined the pattern of androgen receptor expression in the developing urogenital sinus of the intact and neonatally castrated rat in a more direct manner by using antibodies specific to two distinct epitopes of the androgen receptor (14). The use of this immunohistological assay for the presence of the androgen receptor has permitted us to examine specimens prepared from intact rats, rats castrated at birth, and rats castrated at birth and treated with either the
1902
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ANDROGEN RECEPTOR EXPRESSION IN RAT PROSTATE
1903
FIG. 1. A, Three-day-old rat ventral prostate from a noncastrated animal; immunohistological determination using anti-N-terminal antibodies (1:1000 dilution; X150 magnification). B, Ten-dayold rat ventral prostate from a neonatally castrated animal; immunohistological determination using anti-N-terminal antibodies (1:1000 dilution; X150 magnification). C, Ten-day-old rat ventral prostate from an intact animal; immunohistological determination using anti-N-terminal antibodies (1:1000 dilution; X150 magnification). D, Fourteen-day-old rat ventral prostate from a neonatally castrated animal, demonstrating canalization of the prostatic acini; immunohistological determination using anti-N-terminal antibodies (1:1000 dilution; X150 magnification).
antiandrogen flutamide or cortisol to suppress any possible effect of adrenal androgens. These experiments have allowed us to determine whether androgens are required for the developmental pattern of androgen receptor expression that occurs in the postnatal rat prostate. Materials and Methods Cortisol and 5-a-dihydrotestosterone were purchased from Steraloids, Inc. (Wilton, NH), and flutamide was purchased from the Schering Corp. (Union, NJ). All animal experimental protocols were approved by an institutional animal experimentation review board. Seventy newborn male Sprague-Dawley rats (10 pups to 1 nursing mother) were randomly assigned to 1 of 5 groups: intact, castrate, castrate plus dihydrotestosterone (0.01 mg/kg-day), castrate
plus flutamide (50 mg/kg-day), or castrate plus cortisol (10 mg/kg-day). Castration was performed between 12-24 h after birth via an inguinal approach under hypothermia. Both the intact and castrate groups were injected sc at daily intervals with 0.1 ml 2% ethanol in triolein or the same solution containing the androgen antagonist flutamide, cortisol, or dihydrotestosterone. Five animals from each group were killed by decapitation on days 3 and 21 of life, and 1 animal of each group was killed on days 5, 7, 10, and 14. The entire prostate, including the ventral and dorsal lateral lobes, was promptly removed, weighed, placed in OCT compound (Miles Scientific, Naperville, IL), and frozen in a dry ice-acetone bath. Specimens were maintained at -134 C until processed for staining. Affinity-purified polyclonal antibodies that recognize the amino (U402)- or carboxyl (R489)-terminal regions of the rat androgen receptors were used for immunocytochemical evaluations (14). Staining was carried out by the avidin-biotin per-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 September 2015. at 23:11 For personal use only. No other uses without permission. . All rights reserved.
ANDROGEN RECEPTOR EXPRESSION IN RAT PROSTATE
1904
Endo • 1991 Voll28«No4
FIG. 2. Twenty-one-day-old rat ventral prostate; immunohistological determination using anti-N-terminal androgen receptor antibody (1:1000 dilution; X150 magnification) in: intact (noncastrate) rat (A), neonatally castrate rat (B), neonatally castrate rat administered 50 mg/ kg flutamide • day (C), and neonatally castrate rat administered 10 mg/kg cortisol • day.
TABLE 1. Effect of castration and hormone replacement on prostate
weight
Day 3 Day 21
Intact
Castrate
Castrate and flutamide
Castrate and cortisol
Castrate and dihydrotestosterone
20 ± 2 41 ± 3°
18 ± 2 31 ± 2
21 ± 2 30 ± 4
21 ± 2 35 ± 4
23 ± 1 62 ±6*
Three animals from each group were killed and the wet weights of the prostate vere determined. Weight is expressed in milligrams ±SEM. 0 The weight in the intact animal at 21 days was significantly different from that in castrate animals and castrate animals treated with flutamide (P < 0.001). 6 The weight of the prostate tissue from castrate animals treated with dihydrotestosterone was significantly different compared to values in all other groups on day 21 ( P < 0.001).
oxidase method (15) using diaminobenzidine tetrachloride as a chromogen counterstained with 1% hematoxylin. Ten-micron
thick sections of the specimen were cut, fixed in 3.7% paraformaldehyde solution for 10 min, washed in PBS, and incubated at room temperature for 1 h in 4% goat serum. Alternate sections were incubated with either purified anti-N-terminal or anti-Cterminal antiserum diluted at 1:1000 with 3% rat serum. Verification of specific immunoreactivity was established in control experiments by preincubating the affinity-purified antiserum with a 10-fold excess of its respective antigenic peptide at 4 C for 12 h. Primary antibody incubation times were routinely 12 h at 4 C. Statistical evaluations were performed by analysis of variance. P < 0.01 was considered significant.
Results Immunohistological investigations using the anti-Nterminal antibodies revealed the presence of androgen receptors in the stroma of the 3-day-old urogenital sinus,
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 September 2015. at 23:11 For personal use only. No other uses without permission. . All rights reserved.
ANDROGEN RECEPTOR EXPRESSION IN RAT PROSTATE and no androgen receptor immunoreactivity was detected in the prostatic epithelium (see Fig. 1A). The location and apparent intensity of the immunoreactivity detected in the stroma were not altered by neonatal castration (Figs. IB and 2B) or by neonatal castration followed by injections of flutamide (Fig. 2C), cortisol (Fig. 2D), or dihydrotestosterone (data not shown). Similar results were obtained with the anti-C-terminal antibodies (results not shown). To examine the pattern of androgen receptor expression in the neonatal rat prostate, we performed immunohistochemical analyses of the urogenital sinus in animals killed on postnatal days 5, 7, 10, 14, and 21. Immunoreactive androgen receptor was first detectable in the prostatic epithelium on day 10 and was present in these cells thereafter. Having established the normal time course of development of androgen receptor immunoreactivity, we next sought to determine whether this pattern of expression is under androgenic control. Animals were castrated at birth, treated with either vehicle or vehicle containing dihydrotestosterone, flutamide, or cortisol, and killed on postnatal day 5, 7, 10, 14, or 21. Castration and dihydrotestosterone treatment significantly decreased and increased total prostatic weights, respectively, compared to those in intact controls on day 21 (see Table 1). Immunohistochemical and routine histological evaluations with hematoxylin and eosin stains indicate that castration resulted in an increase in the relative amount of mesenchymal and fibrous components of the ventral prostate compared to the epithelium, with fewer prostatic acini being formed; however, no alterations were detected in the androgen receptors of the prostatic epithelium (Figs. 1 and 2). Specifically, similar levels of androgen receptor were present in the prostatic epithelium in the castrated (Fig. IB) and control rats (Fig. 1C) on day 10. On day 14 canalization of the prostate acini was evident in both the neonatally castrate (Fig. ID) and intact rats (data not shown). To remove the effect of residual androgen, androgen receptors were blocked with flutamide (16), or adrenal steroid synthesis was suppressed with exogenous cortisol. Neither regimen altered the pattern of androgen receptor expression (Fig. 2, C and D). That the appearance of androgen receptors in the prostatic epithelium is not dependent on postnatal androgens was also supported by the finding that the administration of supraphysiological doses of dihydrotestosterone from the day of birth did not result in the earlier appearance of epithelial receptors (data not shown).
Discussion The immunohistological data presented here provide direct confirmation of the autoradiographic findings of
1905
prior investigators (7-9), namely that the mesenchyme is the target for androgens during prostatic morphogenesis in the mouse and rat, and the appearance of androgen receptors in the epithelium of the prostate occurs just before or at the same time as establishment of the lumina within the prostatic buds (12). In addition, however, the use of antibody probes that recognize distinctive epitopes of the androgen receptor has permitted an assessment of androgen receptor expression in the absence of ligand, in the presence of antagonists, and in the presence of supraphysiological doses of dihydrotestosterone. Our findings confirm the concept that some aspects of neonatal prostate growth are androgen independent, as had been previously reported by prior investigators (4, 5, 7). Specifically, we concur with Price (4) that formation of lumina occurs within the solid prostatic buds in neonatally castrated rats, a function considered by some to be dependent upon androgens (9). In addition, our data reveal that androgen receptors develop in the prostatic epithelia under diverse conditions of androgen ablation, including neonatal castration and castration plus flutamide or cortisol treatment. These findings suggest that androgens, whether from a testicular or an adrenal source, are not necessary for the development of epithelial receptors in the postnatal rat prostate. In addition, the pattern of expression of the androgen receptor in the prostatic epithelium was not altered when the animal received supraphysiological doses of dihydrotestosterone. These findings suggest that the expression of androgen receptors in the prostatic epithelium is programmed genetically, programmed by androgens before birth, or under the control of other as yet unknown factors.
References 1. Jost A 1970 Hormonal factors in sexual differentiation of the mammalian foetus. Philos Trans R Soc Lond 259:119-130 2. Elger W, Graf K-J, Steinbeck H, Neumann F 1974 Hormonal control of sexual development. Adv Biosci 13:41-69 3. Neumann F, Graf K-J, Elger W 1974 Hormone-induced disturbances in sexual differentiation. Adv Biosci 13:72-101 4. Price D 1936 Normal development of the prostate and seminal vesicles of the rat with a study of experimental postnatal modifications. Am J Anat 60:79-125 5. Lung B, Cunha GR 1981 Development of seminal vesicles and coagulating glands in neonatal mice. The morphologic effects of various hormonal conditions. Anat Rec 199:73-88 6. Chung LWK, Ferland-Raymond G 1975 Differences among rat sex accessory glands in their neonatal androgen dependency. Endocrinology 97:145-153 7. Cunha GR, Donjacour AA, Cooke PJ, Mee S, Bigsby RM, Higgins SJ, Sugimura Y1987 The endocrinology and developmental biology of the prostate. Endocr Rev 8:338-362 8. Shannon JM, Cunha GR 1983 Autoradiographic localization of androgen binding in the developing mouse prostate. Prostate 4:367375 9. Takeda H, Mizmo T, Lasnitzki 11985 Autoradiographic studies of androgen-binding sites in the rat urogenital sinus and postnatal prostate. J Endocrinol 104:87-92 10. Cunha GR, Fuji H, Neubauer BL, Shannon M, Sawyer LM, Reese
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 September 2015. at 23:11 For personal use only. No other uses without permission. . All rights reserved.
ANDROGEN RECEPTOR EXPRESSION IN RAT PROSTATE
1906
BA 1983 Epithelial-mesenchymal interactions in prostatic development. I. Morphological observations of prostatic induction by urogenital sinus mesenchyme in epithelium of the adult rodent urinary bladder. J Cell Biol 96:1662-1670 11. Neubauer BL, Chung LWK, McCormick KA, Taguchi 0, Thompson TC, Cunha GR 1983 Epithelial-mesenchymal interactions in prostatic development. II. Biochemical observations of prostatic induction by urogenital sinus mesenchyme in epithelium of the adult rodent urinary bladder. J Cell Biol 96:1671-1676 12. Cunha GR, Chung LWK, Shannon JM, Taguchi O, Fujii H 1983 Hormone-induced morphogenesis and growth: role of mesenchymal-epithelial interactions. Recent Prog Horm Res 39:559-598 13. Cunha GR, Chung LWK 1981 Stromal-epithelial interactions. I.
Induction of prostatic phenotype in urothelium of testicular feminized (Tfm/Y) mice. J Steroid Biochem 14:1317-1321 14. Husmann DA, Wilson CM, McPhaul MJ, Tilley WD, Wilson JD 1990 Antipeptide antibodies to two distinct regions of the androgen receptor localize the receptor protein to the nuclei of target cells in the rat and human prostate. Endocrinology 126:2359-2368 15. Sar M 1985 Application of avidin-biotin complex technique to the localization of estradiol receptor in target tissues, using monoclonal antibodies. In: Bullock GR, Petruz P (eds) Immunocytochemistry. Academic Press, New York, vol 3:43 16. Neuman F, Elger W 1984 Sexual differentiation: studies with antiandrogens. In: Serio M (ed) Sexual Differentiation: Basic and Clinical Aspects. Raven Press, New York, pp 191-208
Sixteenth European Symposium on Hormones and Cell Regulation September 2 3 - 2 6 , 1991 Mont Ste. Odile, Alsace, France Topics: nitric oxide and cyclic nucleotides, factors in the development of the nervous system, hormonal factors regulating embryonic development, GTP-binding proteins, receptors and channels, role of protein kinases and phosphoprotein phosphatases in signal transduction. Meeting format: invited lectures and posters. Information:
Endo«1991 Voll28«No4
Dr. B. Hamprecht Physiologisch-Chemisches Institut der Universitat Hoppe-Seyler-Str. 4 D-7400 Tubingen, F.R.G. Telfax: 49-7071-293361
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Vol. 128, No. 4 Printed in U.S.A.
Androgen Receptor Expression in the Developing Rat Prostate Is Not Altered by Castration, Flutamide, or Suppression of the Adrenal Axis* DOUGLAS A. HUSMANNf, MICHAEL J. McPHAUL$, AND JEAN D. WILSON Departments of Surgery (Division of Urology) and Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9031
ABSTRACT. Mesenchymal epithelial interactions are believed to be important to the growth and development of the neonatal prostate. Prior studies in the rat ventral prostate, using autoradiography and tritiated dihydrotestosterone, indicate that androgen receptors are present in the prostatic stroma on day 3 and are detected in the epithelium by the tenth postnatal day. These findings suggested that androgen stimulation of the prostatic mesenchyme is a crucial step in the growth and development of the prostate. We have examined this developmental program directly using polyclonal antibodies that recognize specific epitopes of the androgen receptor to examine the pattern of androgen receptor expression in intact and neonatally castrate
M
ORPHOGENESIS of the rat ventral prostate is mediated by androgens, as shown by studies in which androgen ablation (1-3) before sexual differentiation prevents prostate development. In the neonatal period, prostatic growth is also androgen dependent, and castration of neonatal mice or rats inhibits the continued growth and development of the organ (4, 5). Androgens, however, do not appear to be the only factors controlling the growth of the prostate, since neonatal castration (4) or administration of cyproterone acetate (5, 6) does not prevent growth completely. The continued, albeit blunted, development of the prostate after neonatal androgen ablation could have several explanations. First, processes programmed by androgens during embryonic development may continue to operate postnatally in the absence of testicular androgen. Second, adrenal androgens acting upon the androgen receptors (7) may account for the apparent androgen-independent component of Received November 15,1990. Address all correspondence and requests for reprints to: Dr. D. A. Husmann, Division of Urology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 752359031. * This work was supported by grants from the NIH (DK-03892), the Perot Family Foundation, the March of Dimes (Basil O'Connor Award Grant 5-694), and the American Cancer Society (IN-142). t Scholar of the American Urologic Association. X Culpeper Medical Scholar.
animals. In keeping with previous studies, androgen receptors are present in the prostate stroma at birth and subsequently appear in the prostatic epithelium by the 10th postnatal day. Development of androgen receptor expression in the epithelium was not changed when the animals were castrated at birth, castrated and blocked by flutamide, or castrated and given hydrocortisone to suppress the production of adrenal androgens. These findings suggest that the appearance of androgen receptors in the prostatic epithelium is programmed by androgens before birth or that factors other than testicular or adrenal androgens control the development of epithelial androgen receptors. {Endocrinology 128: 1902-1906, 1991)
prostate growth. Finally, other hormones, such as GH, may play an independent role in the growth of the organ. As the action of androgens is mediated via specific intracellular receptors, considerable attention has been focused on the amount and distribution of the androgen receptor in the urogenital sinus and developing prostate. Studies of the stromal-epithelial interactive androgen receptor in the developing prostate have been based on autoradiography using tritiated dihydrotestosterone (713). These studies have demonstrated that the receptor is mesenchymal in location in the prenatal and early postnatal mouse and rat and is detectable within the prostate epithelium at the end of the first postpartum week when the prostatic ducts canalize (8, 9). This functional differentiation in which the receptor develops first in mesenchyme and then in the epithelium is thought to be androgen dependent (9). In the current work we have examined the pattern of androgen receptor expression in the developing urogenital sinus of the intact and neonatally castrated rat in a more direct manner by using antibodies specific to two distinct epitopes of the androgen receptor (14). The use of this immunohistological assay for the presence of the androgen receptor has permitted us to examine specimens prepared from intact rats, rats castrated at birth, and rats castrated at birth and treated with either the
1902
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ANDROGEN RECEPTOR EXPRESSION IN RAT PROSTATE
1903
FIG. 1. A, Three-day-old rat ventral prostate from a noncastrated animal; immunohistological determination using anti-N-terminal antibodies (1:1000 dilution; X150 magnification). B, Ten-dayold rat ventral prostate from a neonatally castrated animal; immunohistological determination using anti-N-terminal antibodies (1:1000 dilution; X150 magnification). C, Ten-day-old rat ventral prostate from an intact animal; immunohistological determination using anti-N-terminal antibodies (1:1000 dilution; X150 magnification). D, Fourteen-day-old rat ventral prostate from a neonatally castrated animal, demonstrating canalization of the prostatic acini; immunohistological determination using anti-N-terminal antibodies (1:1000 dilution; X150 magnification).
antiandrogen flutamide or cortisol to suppress any possible effect of adrenal androgens. These experiments have allowed us to determine whether androgens are required for the developmental pattern of androgen receptor expression that occurs in the postnatal rat prostate. Materials and Methods Cortisol and 5-a-dihydrotestosterone were purchased from Steraloids, Inc. (Wilton, NH), and flutamide was purchased from the Schering Corp. (Union, NJ). All animal experimental protocols were approved by an institutional animal experimentation review board. Seventy newborn male Sprague-Dawley rats (10 pups to 1 nursing mother) were randomly assigned to 1 of 5 groups: intact, castrate, castrate plus dihydrotestosterone (0.01 mg/kg-day), castrate
plus flutamide (50 mg/kg-day), or castrate plus cortisol (10 mg/kg-day). Castration was performed between 12-24 h after birth via an inguinal approach under hypothermia. Both the intact and castrate groups were injected sc at daily intervals with 0.1 ml 2% ethanol in triolein or the same solution containing the androgen antagonist flutamide, cortisol, or dihydrotestosterone. Five animals from each group were killed by decapitation on days 3 and 21 of life, and 1 animal of each group was killed on days 5, 7, 10, and 14. The entire prostate, including the ventral and dorsal lateral lobes, was promptly removed, weighed, placed in OCT compound (Miles Scientific, Naperville, IL), and frozen in a dry ice-acetone bath. Specimens were maintained at -134 C until processed for staining. Affinity-purified polyclonal antibodies that recognize the amino (U402)- or carboxyl (R489)-terminal regions of the rat androgen receptors were used for immunocytochemical evaluations (14). Staining was carried out by the avidin-biotin per-
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ANDROGEN RECEPTOR EXPRESSION IN RAT PROSTATE
1904
Endo • 1991 Voll28«No4
FIG. 2. Twenty-one-day-old rat ventral prostate; immunohistological determination using anti-N-terminal androgen receptor antibody (1:1000 dilution; X150 magnification) in: intact (noncastrate) rat (A), neonatally castrate rat (B), neonatally castrate rat administered 50 mg/ kg flutamide • day (C), and neonatally castrate rat administered 10 mg/kg cortisol • day.
TABLE 1. Effect of castration and hormone replacement on prostate
weight
Day 3 Day 21
Intact
Castrate
Castrate and flutamide
Castrate and cortisol
Castrate and dihydrotestosterone
20 ± 2 41 ± 3°
18 ± 2 31 ± 2
21 ± 2 30 ± 4
21 ± 2 35 ± 4
23 ± 1 62 ±6*
Three animals from each group were killed and the wet weights of the prostate vere determined. Weight is expressed in milligrams ±SEM. 0 The weight in the intact animal at 21 days was significantly different from that in castrate animals and castrate animals treated with flutamide (P < 0.001). 6 The weight of the prostate tissue from castrate animals treated with dihydrotestosterone was significantly different compared to values in all other groups on day 21 ( P < 0.001).
oxidase method (15) using diaminobenzidine tetrachloride as a chromogen counterstained with 1% hematoxylin. Ten-micron
thick sections of the specimen were cut, fixed in 3.7% paraformaldehyde solution for 10 min, washed in PBS, and incubated at room temperature for 1 h in 4% goat serum. Alternate sections were incubated with either purified anti-N-terminal or anti-Cterminal antiserum diluted at 1:1000 with 3% rat serum. Verification of specific immunoreactivity was established in control experiments by preincubating the affinity-purified antiserum with a 10-fold excess of its respective antigenic peptide at 4 C for 12 h. Primary antibody incubation times were routinely 12 h at 4 C. Statistical evaluations were performed by analysis of variance. P < 0.01 was considered significant.
Results Immunohistological investigations using the anti-Nterminal antibodies revealed the presence of androgen receptors in the stroma of the 3-day-old urogenital sinus,
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 September 2015. at 23:11 For personal use only. No other uses without permission. . All rights reserved.
ANDROGEN RECEPTOR EXPRESSION IN RAT PROSTATE and no androgen receptor immunoreactivity was detected in the prostatic epithelium (see Fig. 1A). The location and apparent intensity of the immunoreactivity detected in the stroma were not altered by neonatal castration (Figs. IB and 2B) or by neonatal castration followed by injections of flutamide (Fig. 2C), cortisol (Fig. 2D), or dihydrotestosterone (data not shown). Similar results were obtained with the anti-C-terminal antibodies (results not shown). To examine the pattern of androgen receptor expression in the neonatal rat prostate, we performed immunohistochemical analyses of the urogenital sinus in animals killed on postnatal days 5, 7, 10, 14, and 21. Immunoreactive androgen receptor was first detectable in the prostatic epithelium on day 10 and was present in these cells thereafter. Having established the normal time course of development of androgen receptor immunoreactivity, we next sought to determine whether this pattern of expression is under androgenic control. Animals were castrated at birth, treated with either vehicle or vehicle containing dihydrotestosterone, flutamide, or cortisol, and killed on postnatal day 5, 7, 10, 14, or 21. Castration and dihydrotestosterone treatment significantly decreased and increased total prostatic weights, respectively, compared to those in intact controls on day 21 (see Table 1). Immunohistochemical and routine histological evaluations with hematoxylin and eosin stains indicate that castration resulted in an increase in the relative amount of mesenchymal and fibrous components of the ventral prostate compared to the epithelium, with fewer prostatic acini being formed; however, no alterations were detected in the androgen receptors of the prostatic epithelium (Figs. 1 and 2). Specifically, similar levels of androgen receptor were present in the prostatic epithelium in the castrated (Fig. IB) and control rats (Fig. 1C) on day 10. On day 14 canalization of the prostate acini was evident in both the neonatally castrate (Fig. ID) and intact rats (data not shown). To remove the effect of residual androgen, androgen receptors were blocked with flutamide (16), or adrenal steroid synthesis was suppressed with exogenous cortisol. Neither regimen altered the pattern of androgen receptor expression (Fig. 2, C and D). That the appearance of androgen receptors in the prostatic epithelium is not dependent on postnatal androgens was also supported by the finding that the administration of supraphysiological doses of dihydrotestosterone from the day of birth did not result in the earlier appearance of epithelial receptors (data not shown).
Discussion The immunohistological data presented here provide direct confirmation of the autoradiographic findings of
1905
prior investigators (7-9), namely that the mesenchyme is the target for androgens during prostatic morphogenesis in the mouse and rat, and the appearance of androgen receptors in the epithelium of the prostate occurs just before or at the same time as establishment of the lumina within the prostatic buds (12). In addition, however, the use of antibody probes that recognize distinctive epitopes of the androgen receptor has permitted an assessment of androgen receptor expression in the absence of ligand, in the presence of antagonists, and in the presence of supraphysiological doses of dihydrotestosterone. Our findings confirm the concept that some aspects of neonatal prostate growth are androgen independent, as had been previously reported by prior investigators (4, 5, 7). Specifically, we concur with Price (4) that formation of lumina occurs within the solid prostatic buds in neonatally castrated rats, a function considered by some to be dependent upon androgens (9). In addition, our data reveal that androgen receptors develop in the prostatic epithelia under diverse conditions of androgen ablation, including neonatal castration and castration plus flutamide or cortisol treatment. These findings suggest that androgens, whether from a testicular or an adrenal source, are not necessary for the development of epithelial receptors in the postnatal rat prostate. In addition, the pattern of expression of the androgen receptor in the prostatic epithelium was not altered when the animal received supraphysiological doses of dihydrotestosterone. These findings suggest that the expression of androgen receptors in the prostatic epithelium is programmed genetically, programmed by androgens before birth, or under the control of other as yet unknown factors.
References 1. Jost A 1970 Hormonal factors in sexual differentiation of the mammalian foetus. Philos Trans R Soc Lond 259:119-130 2. Elger W, Graf K-J, Steinbeck H, Neumann F 1974 Hormonal control of sexual development. Adv Biosci 13:41-69 3. Neumann F, Graf K-J, Elger W 1974 Hormone-induced disturbances in sexual differentiation. Adv Biosci 13:72-101 4. Price D 1936 Normal development of the prostate and seminal vesicles of the rat with a study of experimental postnatal modifications. Am J Anat 60:79-125 5. Lung B, Cunha GR 1981 Development of seminal vesicles and coagulating glands in neonatal mice. The morphologic effects of various hormonal conditions. Anat Rec 199:73-88 6. Chung LWK, Ferland-Raymond G 1975 Differences among rat sex accessory glands in their neonatal androgen dependency. Endocrinology 97:145-153 7. Cunha GR, Donjacour AA, Cooke PJ, Mee S, Bigsby RM, Higgins SJ, Sugimura Y1987 The endocrinology and developmental biology of the prostate. Endocr Rev 8:338-362 8. Shannon JM, Cunha GR 1983 Autoradiographic localization of androgen binding in the developing mouse prostate. Prostate 4:367375 9. Takeda H, Mizmo T, Lasnitzki 11985 Autoradiographic studies of androgen-binding sites in the rat urogenital sinus and postnatal prostate. J Endocrinol 104:87-92 10. Cunha GR, Fuji H, Neubauer BL, Shannon M, Sawyer LM, Reese
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 September 2015. at 23:11 For personal use only. No other uses without permission. . All rights reserved.
ANDROGEN RECEPTOR EXPRESSION IN RAT PROSTATE
1906
BA 1983 Epithelial-mesenchymal interactions in prostatic development. I. Morphological observations of prostatic induction by urogenital sinus mesenchyme in epithelium of the adult rodent urinary bladder. J Cell Biol 96:1662-1670 11. Neubauer BL, Chung LWK, McCormick KA, Taguchi 0, Thompson TC, Cunha GR 1983 Epithelial-mesenchymal interactions in prostatic development. II. Biochemical observations of prostatic induction by urogenital sinus mesenchyme in epithelium of the adult rodent urinary bladder. J Cell Biol 96:1671-1676 12. Cunha GR, Chung LWK, Shannon JM, Taguchi O, Fujii H 1983 Hormone-induced morphogenesis and growth: role of mesenchymal-epithelial interactions. Recent Prog Horm Res 39:559-598 13. Cunha GR, Chung LWK 1981 Stromal-epithelial interactions. I.
Induction of prostatic phenotype in urothelium of testicular feminized (Tfm/Y) mice. J Steroid Biochem 14:1317-1321 14. Husmann DA, Wilson CM, McPhaul MJ, Tilley WD, Wilson JD 1990 Antipeptide antibodies to two distinct regions of the androgen receptor localize the receptor protein to the nuclei of target cells in the rat and human prostate. Endocrinology 126:2359-2368 15. Sar M 1985 Application of avidin-biotin complex technique to the localization of estradiol receptor in target tissues, using monoclonal antibodies. In: Bullock GR, Petruz P (eds) Immunocytochemistry. Academic Press, New York, vol 3:43 16. Neuman F, Elger W 1984 Sexual differentiation: studies with antiandrogens. In: Serio M (ed) Sexual Differentiation: Basic and Clinical Aspects. Raven Press, New York, pp 191-208
Sixteenth European Symposium on Hormones and Cell Regulation September 2 3 - 2 6 , 1991 Mont Ste. Odile, Alsace, France Topics: nitric oxide and cyclic nucleotides, factors in the development of the nervous system, hormonal factors regulating embryonic development, GTP-binding proteins, receptors and channels, role of protein kinases and phosphoprotein phosphatases in signal transduction. Meeting format: invited lectures and posters. Information:
Endo«1991 Voll28«No4
Dr. B. Hamprecht Physiologisch-Chemisches Institut der Universitat Hoppe-Seyler-Str. 4 D-7400 Tubingen, F.R.G. Telfax: 49-7071-293361
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