ANDROGEN RECEPTORS IN THE RAT BRAIN, ANTERIOR PITUITARY GLAND AND VENTRAL PROSTATE GLAND:
EFFECTS OF ORCHIDECTOMY AND AGEING B. D. GREENSTEIN
Department of Pharmacology, Chelsea College, London SW3 6LX
(Received 3 July 1978) SUMMARY
Available high-affinity binding sites for 5\g=a\-dihydrotestosterone (DHT) were measured in cytosols obtained from the amygdala, hypothalamus, anterior pituitary gland and ventral prostate gland of 12-week-old rats at various times after orchidectomy, and in the corresponding tissues of 18-month-old male rats. It is suggested that the lower affinity of the DHT binding reaction in brain and ventral prostatic cytosols after orchidectomy or ageing respectively, may explain, at least in part, the changes in the responsiveness of the tissues to
androgens.
INTRODUCTION
Macromolecules which bind dihydrotestosterone (DHT) with high affinity occur in cytosols prepared from the ventral prostate gland, anterior pituitary gland and discrete areas of the brain of rats orchidectomized for 3 days (Ginsburg, Greenstein, MacLusky, Morris &
Thomas, 1974a; Barley, Ginsburg, Greenstein, MacLusky & Thomas, 1975; Kato, 1975; Naess, Attramadal & Aakvaag, 1975; Naess, Hansson, Djoeseland & Attramadal, 1975; Thieulant, Mercier, Samperez & Jouan, 1975). In most studies of androgen receptors in
adult rats the animals were orchidectomized at least 18 h before death. This was done to reduce the levels of endogenous androgens. It has been previously reported (Barley et al. 1975) that no high-affinity binding of DHT is detectable in the brain or pituitary gland of intact adult male rats, but that binding is apparent 3 days after orchidectomy. Since serum concentrations of testosterone are negligible within 24 h after orchidectomy of the adult rat (Coyotupa, Parlow & Kovacic, 1973), it was considered of interest to investigate the effects of orchidectomy on the binding of DHT in more detail. In addition, the parameters of high-affinity DHT binding have been measured in the tissues of 18-month-old male rats. Some of this work has been reported previously in preliminary form (Ginsburg & Green¬
stein, 1976, 1977).
MATERIALS AND METHODS
Animals
study of the effects of orchidectomy on androgen receptors, 12-week-old rats were orchidectomized under ether anaesthesia through the scrotal route and then maintained as described elsewhere (Barley et al. 1975). Animals described below as 'intact' were shamoperated by making an incision in the scrotum but the testes were not removed. At selected times after the operation the animals were decapitated and cytosols prepared. To study the effects of ageing, 18-month-old rats were orchidectomized or sham operated on. * Present address : Department of Pharmacology, St Thomas's Hospital Medical School, London, SEI 7EH.
For the
The materials and methods used for the
preparation
of
cytosolic fractions
and the
measurement of available high-affinity sites for DHT in vitro have been described elsewhere (Barley et al. 1975), as have the procedures for the measurement of total protein content
and radioactivity from five animals
(Ginsburg, Greenstein, MacLusky, Morris & Thomas, 19746). were pooled in order to prepare the cytosols.
Tissues
Assay A range of concentrations of [3H]DHT (47 Ci/mmol; Radiochemical Centre, Amersham) were prepared in buffer (phosphate, 0-01 mol/1; sucrose, 0-25 mol/1; pH 7-4). A portion of each solution (0-1 ml) was added to 0-2 ml cytosol at 0 °C to give final concentrations of [3H]DHT in the incubate of 0-3 10-»-6 10~9 mol/1 (9390-187 812 disintegrations min-1 incubate-1). A parallel set of incubates contained, in addition, excess unlabelled DHT (6 10~7 mol/1). After 2 h of incubation at 0 °C, portions of the incubates (0-2 ml) were layered on small columns of Sephadex LH-20 (0-45 9 cm) at 0-5—1CC and washed in with 0-1 ml buffer. After 1 h of stopped flow, the bound radioactivity was eluted in 0-7 ml buffer into scin¬ tillation vials and the bound eluted radioactivity was measured. The saturable bound counts/ min were obtained by difference after construction of binding isotherms (Barley et al. 1975), and the dissociation constant (KA) and binding capacity calculated from the linear Scatchard plots were obtained. Experiments on each tissue were repeated up to six times in some cases. The data from several Scatchard plots can be compared on the same graph by modifying the Scatchard (1949) plot (Ginsburg & Greenstein, 1977). If bound hormone/free hormone n.Ka binding capacity and K& molar associ¬ . bound hormone, where ation constant (1/mol), is divided throughout by n, then bound hormone/(n. free hormone) ^a—#a-(bound hormone/n). If bound hormone/(n. free hormone) is plotted against bound hormone/n, then the linear Scatchard plot should intersect the abscissa at unity. =
=
=
—
=
RESULTS
Effects of orchidectomy on 12-week-old rats of High-affinity binding DHT could not be detected in cytosol from the anterior pituitary gland, the neocortex, hypothalamus or amygdala of intact 12-week-old male rats; the binding was measurable, however, in cytosol from the ventral prostate gland (Table 1). However, after orchidectomy high-affinity binding of DHT could be measured in cytosol from the brain and anterior pituitary gland. The abundance of binding sites in all neural Table 1.
on the numbers of high-affinity receptors for [3H]dihydrocomponents of rat brain and the anterior pituitary and ventral prostate
Effect of orchidectomy
testosterone in various
glands of 12-week-old rats (means + S.E.M.; numbers of receptors/mg cytosol protein 10~9*; numbers in parentheses indicate numbers of experiments. Tissues fromfive animals were pooled in each experiment) Time after orchidectomy (days) Tissue Neocortex
0 ND ND
0-75 5-4 ±0-3 4-9 ±0-6
7
21
4-4 + 0-4(5) (5) 5-2±l-0(3) 5-3 ±0-9 (4) 5-9 ±0-7 (6) Amygdala (4) ND 6-3 + 0-7(6) Hypothalamus 5-5±0-8(4) 5-4±l-0(5) Anterior pituitary gland ND 71 ±1-3 (3) 7-0±0-6(4) 10-5±l-2(5) Ventral prostate gland 15-6(2) 37-6±7-0(5) 22-6±2-2(5) 12-l±3-9(4) * Obtained by multiplying the molar binding capacity by Avogadro's number. ND, Not detectable. Anterior pituitary gland: 0-75 v. 56 days, /'
EFFECTS OF ORCHIDECTOMY AND AGEING B. D. GREENSTEIN
Department of Pharmacology, Chelsea College, London SW3 6LX
(Received 3 July 1978) SUMMARY
Available high-affinity binding sites for 5\g=a\-dihydrotestosterone (DHT) were measured in cytosols obtained from the amygdala, hypothalamus, anterior pituitary gland and ventral prostate gland of 12-week-old rats at various times after orchidectomy, and in the corresponding tissues of 18-month-old male rats. It is suggested that the lower affinity of the DHT binding reaction in brain and ventral prostatic cytosols after orchidectomy or ageing respectively, may explain, at least in part, the changes in the responsiveness of the tissues to
androgens.
INTRODUCTION
Macromolecules which bind dihydrotestosterone (DHT) with high affinity occur in cytosols prepared from the ventral prostate gland, anterior pituitary gland and discrete areas of the brain of rats orchidectomized for 3 days (Ginsburg, Greenstein, MacLusky, Morris &
Thomas, 1974a; Barley, Ginsburg, Greenstein, MacLusky & Thomas, 1975; Kato, 1975; Naess, Attramadal & Aakvaag, 1975; Naess, Hansson, Djoeseland & Attramadal, 1975; Thieulant, Mercier, Samperez & Jouan, 1975). In most studies of androgen receptors in
adult rats the animals were orchidectomized at least 18 h before death. This was done to reduce the levels of endogenous androgens. It has been previously reported (Barley et al. 1975) that no high-affinity binding of DHT is detectable in the brain or pituitary gland of intact adult male rats, but that binding is apparent 3 days after orchidectomy. Since serum concentrations of testosterone are negligible within 24 h after orchidectomy of the adult rat (Coyotupa, Parlow & Kovacic, 1973), it was considered of interest to investigate the effects of orchidectomy on the binding of DHT in more detail. In addition, the parameters of high-affinity DHT binding have been measured in the tissues of 18-month-old male rats. Some of this work has been reported previously in preliminary form (Ginsburg & Green¬
stein, 1976, 1977).
MATERIALS AND METHODS
Animals
study of the effects of orchidectomy on androgen receptors, 12-week-old rats were orchidectomized under ether anaesthesia through the scrotal route and then maintained as described elsewhere (Barley et al. 1975). Animals described below as 'intact' were shamoperated by making an incision in the scrotum but the testes were not removed. At selected times after the operation the animals were decapitated and cytosols prepared. To study the effects of ageing, 18-month-old rats were orchidectomized or sham operated on. * Present address : Department of Pharmacology, St Thomas's Hospital Medical School, London, SEI 7EH.
For the
The materials and methods used for the
preparation
of
cytosolic fractions
and the
measurement of available high-affinity sites for DHT in vitro have been described elsewhere (Barley et al. 1975), as have the procedures for the measurement of total protein content
and radioactivity from five animals
(Ginsburg, Greenstein, MacLusky, Morris & Thomas, 19746). were pooled in order to prepare the cytosols.
Tissues
Assay A range of concentrations of [3H]DHT (47 Ci/mmol; Radiochemical Centre, Amersham) were prepared in buffer (phosphate, 0-01 mol/1; sucrose, 0-25 mol/1; pH 7-4). A portion of each solution (0-1 ml) was added to 0-2 ml cytosol at 0 °C to give final concentrations of [3H]DHT in the incubate of 0-3 10-»-6 10~9 mol/1 (9390-187 812 disintegrations min-1 incubate-1). A parallel set of incubates contained, in addition, excess unlabelled DHT (6 10~7 mol/1). After 2 h of incubation at 0 °C, portions of the incubates (0-2 ml) were layered on small columns of Sephadex LH-20 (0-45 9 cm) at 0-5—1CC and washed in with 0-1 ml buffer. After 1 h of stopped flow, the bound radioactivity was eluted in 0-7 ml buffer into scin¬ tillation vials and the bound eluted radioactivity was measured. The saturable bound counts/ min were obtained by difference after construction of binding isotherms (Barley et al. 1975), and the dissociation constant (KA) and binding capacity calculated from the linear Scatchard plots were obtained. Experiments on each tissue were repeated up to six times in some cases. The data from several Scatchard plots can be compared on the same graph by modifying the Scatchard (1949) plot (Ginsburg & Greenstein, 1977). If bound hormone/free hormone n.Ka binding capacity and K& molar associ¬ . bound hormone, where ation constant (1/mol), is divided throughout by n, then bound hormone/(n. free hormone) ^a—#a-(bound hormone/n). If bound hormone/(n. free hormone) is plotted against bound hormone/n, then the linear Scatchard plot should intersect the abscissa at unity. =
=
=
—
=
RESULTS
Effects of orchidectomy on 12-week-old rats of High-affinity binding DHT could not be detected in cytosol from the anterior pituitary gland, the neocortex, hypothalamus or amygdala of intact 12-week-old male rats; the binding was measurable, however, in cytosol from the ventral prostate gland (Table 1). However, after orchidectomy high-affinity binding of DHT could be measured in cytosol from the brain and anterior pituitary gland. The abundance of binding sites in all neural Table 1.
on the numbers of high-affinity receptors for [3H]dihydrocomponents of rat brain and the anterior pituitary and ventral prostate
Effect of orchidectomy
testosterone in various
glands of 12-week-old rats (means + S.E.M.; numbers of receptors/mg cytosol protein 10~9*; numbers in parentheses indicate numbers of experiments. Tissues fromfive animals were pooled in each experiment) Time after orchidectomy (days) Tissue Neocortex
0 ND ND
0-75 5-4 ±0-3 4-9 ±0-6
7
21
4-4 + 0-4(5) (5) 5-2±l-0(3) 5-3 ±0-9 (4) 5-9 ±0-7 (6) Amygdala (4) ND 6-3 + 0-7(6) Hypothalamus 5-5±0-8(4) 5-4±l-0(5) Anterior pituitary gland ND 71 ±1-3 (3) 7-0±0-6(4) 10-5±l-2(5) Ventral prostate gland 15-6(2) 37-6±7-0(5) 22-6±2-2(5) 12-l±3-9(4) * Obtained by multiplying the molar binding capacity by Avogadro's number. ND, Not detectable. Anterior pituitary gland: 0-75 v. 56 days, /'
