0021-972X/92/7501-0308$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright 0 1992 by The Endocrine Society
Vol. 75, No. 1 Printed in U.S.A.
Steroid Hormone Receptors in the Adrenal Fetal and Adult Rhesus Monkeys* JONATHAN J. HIRST, MILES J. NOVY
NEAL
B. WEST+,
ROBERT
M. BRENNER,
Glands
of
AND
Division of Reproductive Biology and Behavior (J.J.H., N.B. W., R.M.B., M.J.N.), Oregon Regional Primate Research Center, Beaverton, Oregon 97006; and the Department of Obstetrics and Gynecology (M. J.N.), Oregon Health Sciences University, Portland, Oregon 97201 ABSTRACT Sex steroid hormone receptors have been identified in the adrenal glands of rodents and may have a role in adrenal function. The highly estrogenic environment during pregnancy has been proposed to influence steroidogenesis by the fetal zone of the primate fetal adrenal gland. In order to determine whether these effects involve receptormediated mechanisms, we have examined the concentration and distribution of estrogen receptor (ER), androgen receptor, and progesterone receptor (PR) in the adrenal glands of fetal, immature, and adult rhesus monkeys. Monoclonal antibodies were used for immunocytochemistry (ICC), and in a gradient shift assay, for determination of receptor distribution and concentration, respectively. There was no difference between the concentrations of ER in the adrenal glands from male and female adult animals (12.4 f 2.2, n = 3 and 13.2 + 2.0 fmol/mg DNA, n = 7; respectively); however, the concentration of ER in the fetal adrenal glands was markedly lower than in the adults (combined adult 12.7 + 1.6, n = 10, and fetal 0.9 f 0.4 fmol/mg DNA,
n = 7; P < 0.01). The concentration of ER in the adrenal glands of immature animals was also lower compared to adult animals (6.1 f 1.6, n = 6, P < 0.05). In the adult, ICC revealed that staining for ER was restricted to the cell nucleus and was most dense in the zona fasciculata, with lesser staining in the zona glomerulosa and zona reticularis, and with no detectable staining in the medulla. ER staining was virtually absent in the fetal zone which comprises the major portion of the fetal gland; however, some staining was observed in the narrow definitive zone. The distribution of androgen receptor was similar to that of ER, whereas there was no detectable staining for PR in the adrenals of either adult or fetal animals. We conclude: 1) that the lower concentration of ER in fetal adrenal glands is due to the absence of ER in the fetal zone; 2) the lack of ER and PR in the fetal zone suggests that estrogens and progestins do not influence the growth or function of the fetal zone by receptor-mediated mechanisms; 3) estrogens and androgens may influence the function of the adult adrenal cortex. (J Clin Endocrinol Metab 75: 30%314,1992)
S
is inhibited by estrogens during midgestation in baboons, suggesting that estrogens may reduce the responsivenessof the fetal adrenal to ACTH. The putative variable effects of estrogens on adrenal function may involve changes in the concentration and distribution of estrogen receptors (ER). The present studies were undertaken to determine whether the reported effects of sex steroid hormones on the primate adrenal gland involve receptor-mediated mechanismsand to determine the timing of receptor development in the fetal adrenal glands. Therefore, we examined: 1) the concentration and distribution of ER, AR, and progesterone receptor (PR) in the adrenal glands of adult rhesus monkeys; 2) the concentrations and distribution of ER in midgestation and nearterm fetuses and immature monkeys.
pecific high affinity estrogen binding has been identified in the adrenal glands of adult rats, and steroid hormones have been shown to influence adrenal steroidogenesis(l-3). Androgen treatment has also been reported to influence adrenal function and the presence of androgen receptors (AR) in rodent speciessuggeststhat the effect of androgens involves receptor-mediated pathways (4, 5). The fetal zone of the primate fetal adrenal glands synthesizesand secretesandrogen precursors for placental estrogen production and is responsible for the highly estrogenic environment during late gestation (6-8). ACTH is established as the principal regulator of androgen production by the fetal adrenal (9); however, estrogenshave been proposed to have a role in the suppressionof 3P-hydroxysteroid dehydrogenases (3PHSD) levels in the fetal zone. This action would reduce fetal cortisol concentrations leading to elevated dehydroepiandrosterone (DHEA) secretion by the fetal zone (10). Alternatively, Albrecht and Pepe (11) have reported that ACTH-stimulated DHEA production by fetal zone cells
Subjects Collection
of
adrenal
and Methods
glands
Rhesus monkey fetuses between 127 and 160 days of gestational age (term 167 days) were obtained by cesarean section at the Oregon Regional Primate Research Center (ORI’RC). Cesarean section was performed under Halothane/nitrous oxide anesthesia and fetal adrenal glands were obtained within 30 min of removal of the fetus. Adrenal glands from neonatal, immature, and adult animals were obtained at autopsies conducted as part of the ORPRC tissue distribution program. These animals were killed by overdose with pentobarbitone sodium followed by exsanguination and the adrenal tissue was obtained within 30 min of the death of the animals. All adrenal glands were transported to our laboratories in Hanks’ balanced salt solution at 20 C for receptor
Received August 13, 199 1. Address all correspondence and reprint requests to: Jonathan J. Hirst, Ph.D., Oregon Regional Primate Research Center, 505 N.W. 185th Avenue, Beaverton, Oregon 97006. *This work was supported by NIH Grants RR-00163, HD-06159 (M.J.N.) and HD-19182 (R.M.B.). Publication No. 1837 of the Oregon Regional Primate Research Center. t Current address: Office of Extramural Programs, Building 31, Room 5B35, National Institutes of Health, Bethesda, Maryland 20892.
308
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 02 March 2015. at 18:45 For personal use only. No other uses without permission. . All rights reserved.
ADRENAL
STEROID
assay or immunocytochemical (ICC) studies. Blood samples were tained from all female animals at the time of autopsy or surgery determination of plasma 17P-estradiol concentration by RIA which previously been described (12).
HORMONE obfor has
RECEPTORS
309
radioactivity in the peak was taken as the amount of specific receptor. Receptor concentration values were expressed as fmol 3H-Ez per mg DNA in the KC1 extracted pellet.
Immunocytochemistry Monoclonal
antibodies
The monoclonal anti-ER (H-222) was raised against purified human MCF-7 cell extranuclear ER‘and was provided by Abbott Laboratories (North Chicaeo. IL). The PR monoclonal antibodv fB39) was urovided by Dr. G. Gyeene’(University of Chicago) and’the AR mdnoclonal antibody (ANl-15) was provided by Dr. S. Liao (University of Chicago). A nonspecific control antibody of the same immunoglobulin subclass, raised against Timothy grass pollen antigen B (AT) was provided Dr. A. Malley (ORPRC).
Estrogen
and androgen
receptor assays
We utilized a gradient shift assay, as previously described (13-15) to measure ER and AR in adrenal tissue. The assay involves the incubation of tritiated ligand with fresh tissue at 37 C and the use of specific
monoclonal
antibodies to separate receptor-bound
ligand from free
ligand on sucrose gradients. The advantage of this assay is that low levels of receptors in small samples can be reliably quantified because the specific antibody shifts the labeled receptors away from nonspecifi-
callv labeled molecules and free liaand on the gradient. This facilitates discrimination between specific aid nonspecifically bound ligand and avoids the use of absorbants which may reduce specific binding, thereby leading to an underestimation of receptor concentrations. A series of preliminary studies were conducted to determine the optimal assay conditions for adrenal tissue and these studies are described in Results. Subsequently all assays were performed by the following procedure. Adrenal gland tissue from adult, immature, and neonatal animals was cut into slices of 0.5 mm thickness with a Stadie-Riggs tissue slicer (Arthur H. Thomas Co., Philadelphia, PA). Fetal adrenal tissue was chopped into cubes 0.5-1.0 mm in diameter. The adrenal tissue was rinsed in Trowell’s medium, containing 0.7% (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid), pH 7.0, at 37 C and then incubated in the same medium with the addition of 10 nmol tritiated 17P-estradiol (3H-E2) or tritiated 5a-dihydrotestosterone (NEN DuPont, Wilmington, DE). The incubations were of 60-min duration and were conducted
Slices of -2 mm thickness were cut from adrenal glands in a crosssectional plane extending from the anteromedial apex to the center of the renal depression as described by McNulty et al. (17). The tissue was imbedded in a drop of Tissue-Tek OTC (Miles Laboratories, Naperville, IL) and frozen by plunging in liquid propane (-130 C). The sections were stored over liquid nitrogen until processed further. Cryostat sections (4-6 Frn) were cut at -20 C on a Hacker-Bright cryostat (Hacker Instruments, Fairfield, NJ) and thaw mounted on gelatin-coated glass slides. Tissue sections were prepared for ICC as described by McClellan et aI. (18) using the indirect method with avitin-biotin complex except for the following modifications. Frozen tissue sections were fixed at 4 C in picric acid paraformaldehyde for lo-12 min and then 85% ethanol for 4 min. The slides were rinsed in phosphate-buffered saline (PBS), pH 7.4, placed in sodium borohydride for 4 min, and rinsed again with PBS containing 1% gelatin. Next the tissue sections were incubated with normal rabbit serum for 30 min followed by an incubation with monoclonal anti-ER, PR, AR, or AT. The incubations were conducted at 4 C, in a moist chamber, overnight. The following day the sections were rinsed with PBS and incubated with normal rabbit serum and then with biotinylated anti-rat immunoglobulin G for 30 min at 20 C. The biotin was detected with an avitin-biotin peroxidase kit (Vector Laboratories, Burlingame, CA). All slides that were compared for staining intensity were processed in the same ICC run on the same day and color photomicrographs were taken with planapochromatic lenses on Ektar 25 film.
Statistical
analysis
Biochemical data were analyzed by Student’s t test and, when more than two groups were considered, by one-way analysis of variance followed by Duncan’s multiple range test. Correlation was determined by least squares method.
Results
under an atmosphere of 60% 02, 34% N2, 6% COZ. At the conclusion of the incubations the tissue was frozen and stored over liquid nitrogen prior to further processing. There was no difference in the concentration of ER detected when the tissue was frozen at this stage compared to immediate processing. Frozen adrenal tissue was homogenized in TEDGM buffer (10 mmol Tris, 1.5 mmol ethylenediaminetetraacetic acid [EDTA], 1 mmol dithiotreitol [DDT], 10% vol/vol glycerol, 10 mmol sodium molybdate, pH 7.4) at the rate of 100 mg tissue/ml at 4 C with a glass/glass hand homogenizer. The homogenate was centrifuged at 2,500 X g for 10 min, at 4 C, vielding a crude nuclear pellet, The supernatant was retained as the cytdsolic fraction. The pellet-was washed ihree times with the same volume of TEDGM and the final uellet was resusuended in TEDK buffer (10 mmol Tris, 1.5 mmol EDTAt 1 mmol dithiotreitol, 0.5 mmol KCl, pH 7.4; 200 mg original tissue/l mL) to extract nuclear receptor. Extrac-
tions were for 60 min at 0 C with gentle agitation at 15-min intervals. Following nuclear extraction the suspension was centrifuged at 10,000 x g for 10 min. The pellet was stored at -20 C, prior to assay for DNA (16), and the supernatant was divided into 500 PL aliquots. Each aliquot was added to 2.5 Kg of monoclonal antibody, allowed to incubate for 60 min at 0 C, and then subjected to sedimentation analysis on 5-20% sucrose gradients (prepared in 10 mmol Tris, 1.5 mmol EDTA, 0.5 KCl). Protein solutions (BSA, 4.5s and aldolase, 8S), that were used as sedimentation markers, were layered and run on a parallel gradient. The gradients were centrifuged at 205,000 x g for 18 h at 4 C. Following fractionation of the gradients, each fraction was mixed with Scintiverse BD (Fisher Scientific, Pittsburgh, PA) and radioactivity was quantitated with a model 460-CD Packard liquid scintillation counter. The radioactivity associated with the 8s shifted peak of the gradient profile resulted
from tritiated steroid-receptor
antibody complexes and the quantity of
Steroid
receptor assays
The major assay parameters were investigated in preliminary experiments at least twice. The monoclonal antibodies formed complexes with 3H-E,-labeled receptors and these complexes were detected as peaks, observed in the 8s position, on sucrose gradients (Fig. 1). These peaks were shifted away from free 3H-E2 and nonspecifically bound 3H-E2 at the top of the gradient. As shown in Fig. 1, the 8s peak was absent when a nonspecific antibody of the same immunoglobulin subclass (AT), or when the PR antibody was used in place of the anti-ER under the same conditions with the same tissue. Thus, the labeled ER remained near the top of the gradient and obscured by the presence of free 3H-E2 unless the ER monoclonal antibody was present. The incubation of adrenal tissue slices with *H-E3 at 37 C for increasing time periods resulted in maximal labeling and detection of nuclear ER at 60 min of incubation (Fig. 2). Incubations of 60 min were therefore used for all further studies. There was a slight decrease in ER detection when incubations were extended to 120 min, suggesting that receptor processing and/or degradation may occur at 37 C with longer incubations. Optimal extraction of nuclear ER was obtained with 60 min of extraction at 0 C and with 0.5 mol KC1 as the
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 02 March 2015. at 18:45 For personal use only. No other uses without permission. . All rights reserved.
HIRST ET AL.
310
FIG. 1. Sucrose density gradient profile of 3H-E2 binding in nuclear extracts of adrenal tissue from rhesus monkeys. Slices of fresh tissue were incubated with 10 nmol/L 3H-E, and nuclear extracts of the tissue were mixed with monoclonal antibodies, anti-ER, anti-PR, and with a nonspecific control antibody (antiTimothy grass pollen antigen B, AT) and run on 5-20% sucrose gradient run for 18 h at 205,600 x g. The shaded area indicates the peak resulting from the sedimentation 3H-Ez labeled receptorantibody complexes in the presence of anti-ER. The quantity of ‘H-E2 associated with this peak (shaded area) was used as a measure of ER. No peaks were observed with either anti-PR or AT. The positions of BSA (4.5s) and aldolase (8s) marker proteins are indicated.
- -
Anti-E!?
-.-
Anti-PI?
-
Control
JCE & M. 1992 Vol75.Nol
A%!~e
BSA 4.5s
4
z !r 11 I ‘i
Receptor
Antibody
l,,,,,,,,J,,,,,,,,I
1
4
6
8
10
12
14
16
18
20
I
/,
22
I,
24
Botto:
26 TOD
Fraction
Number
12
14r
1
10
Incubation
30
Time
60
I
Vol. 75, No. 1 Printed in U.S.A.
Steroid Hormone Receptors in the Adrenal Fetal and Adult Rhesus Monkeys* JONATHAN J. HIRST, MILES J. NOVY
NEAL
B. WEST+,
ROBERT
M. BRENNER,
Glands
of
AND
Division of Reproductive Biology and Behavior (J.J.H., N.B. W., R.M.B., M.J.N.), Oregon Regional Primate Research Center, Beaverton, Oregon 97006; and the Department of Obstetrics and Gynecology (M. J.N.), Oregon Health Sciences University, Portland, Oregon 97201 ABSTRACT Sex steroid hormone receptors have been identified in the adrenal glands of rodents and may have a role in adrenal function. The highly estrogenic environment during pregnancy has been proposed to influence steroidogenesis by the fetal zone of the primate fetal adrenal gland. In order to determine whether these effects involve receptormediated mechanisms, we have examined the concentration and distribution of estrogen receptor (ER), androgen receptor, and progesterone receptor (PR) in the adrenal glands of fetal, immature, and adult rhesus monkeys. Monoclonal antibodies were used for immunocytochemistry (ICC), and in a gradient shift assay, for determination of receptor distribution and concentration, respectively. There was no difference between the concentrations of ER in the adrenal glands from male and female adult animals (12.4 f 2.2, n = 3 and 13.2 + 2.0 fmol/mg DNA, n = 7; respectively); however, the concentration of ER in the fetal adrenal glands was markedly lower than in the adults (combined adult 12.7 + 1.6, n = 10, and fetal 0.9 f 0.4 fmol/mg DNA,
n = 7; P < 0.01). The concentration of ER in the adrenal glands of immature animals was also lower compared to adult animals (6.1 f 1.6, n = 6, P < 0.05). In the adult, ICC revealed that staining for ER was restricted to the cell nucleus and was most dense in the zona fasciculata, with lesser staining in the zona glomerulosa and zona reticularis, and with no detectable staining in the medulla. ER staining was virtually absent in the fetal zone which comprises the major portion of the fetal gland; however, some staining was observed in the narrow definitive zone. The distribution of androgen receptor was similar to that of ER, whereas there was no detectable staining for PR in the adrenals of either adult or fetal animals. We conclude: 1) that the lower concentration of ER in fetal adrenal glands is due to the absence of ER in the fetal zone; 2) the lack of ER and PR in the fetal zone suggests that estrogens and progestins do not influence the growth or function of the fetal zone by receptor-mediated mechanisms; 3) estrogens and androgens may influence the function of the adult adrenal cortex. (J Clin Endocrinol Metab 75: 30%314,1992)
S
is inhibited by estrogens during midgestation in baboons, suggesting that estrogens may reduce the responsivenessof the fetal adrenal to ACTH. The putative variable effects of estrogens on adrenal function may involve changes in the concentration and distribution of estrogen receptors (ER). The present studies were undertaken to determine whether the reported effects of sex steroid hormones on the primate adrenal gland involve receptor-mediated mechanismsand to determine the timing of receptor development in the fetal adrenal glands. Therefore, we examined: 1) the concentration and distribution of ER, AR, and progesterone receptor (PR) in the adrenal glands of adult rhesus monkeys; 2) the concentrations and distribution of ER in midgestation and nearterm fetuses and immature monkeys.
pecific high affinity estrogen binding has been identified in the adrenal glands of adult rats, and steroid hormones have been shown to influence adrenal steroidogenesis(l-3). Androgen treatment has also been reported to influence adrenal function and the presence of androgen receptors (AR) in rodent speciessuggeststhat the effect of androgens involves receptor-mediated pathways (4, 5). The fetal zone of the primate fetal adrenal glands synthesizesand secretesandrogen precursors for placental estrogen production and is responsible for the highly estrogenic environment during late gestation (6-8). ACTH is established as the principal regulator of androgen production by the fetal adrenal (9); however, estrogenshave been proposed to have a role in the suppressionof 3P-hydroxysteroid dehydrogenases (3PHSD) levels in the fetal zone. This action would reduce fetal cortisol concentrations leading to elevated dehydroepiandrosterone (DHEA) secretion by the fetal zone (10). Alternatively, Albrecht and Pepe (11) have reported that ACTH-stimulated DHEA production by fetal zone cells
Subjects Collection
of
adrenal
and Methods
glands
Rhesus monkey fetuses between 127 and 160 days of gestational age (term 167 days) were obtained by cesarean section at the Oregon Regional Primate Research Center (ORI’RC). Cesarean section was performed under Halothane/nitrous oxide anesthesia and fetal adrenal glands were obtained within 30 min of removal of the fetus. Adrenal glands from neonatal, immature, and adult animals were obtained at autopsies conducted as part of the ORPRC tissue distribution program. These animals were killed by overdose with pentobarbitone sodium followed by exsanguination and the adrenal tissue was obtained within 30 min of the death of the animals. All adrenal glands were transported to our laboratories in Hanks’ balanced salt solution at 20 C for receptor
Received August 13, 199 1. Address all correspondence and reprint requests to: Jonathan J. Hirst, Ph.D., Oregon Regional Primate Research Center, 505 N.W. 185th Avenue, Beaverton, Oregon 97006. *This work was supported by NIH Grants RR-00163, HD-06159 (M.J.N.) and HD-19182 (R.M.B.). Publication No. 1837 of the Oregon Regional Primate Research Center. t Current address: Office of Extramural Programs, Building 31, Room 5B35, National Institutes of Health, Bethesda, Maryland 20892.
308
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 02 March 2015. at 18:45 For personal use only. No other uses without permission. . All rights reserved.
ADRENAL
STEROID
assay or immunocytochemical (ICC) studies. Blood samples were tained from all female animals at the time of autopsy or surgery determination of plasma 17P-estradiol concentration by RIA which previously been described (12).
HORMONE obfor has
RECEPTORS
309
radioactivity in the peak was taken as the amount of specific receptor. Receptor concentration values were expressed as fmol 3H-Ez per mg DNA in the KC1 extracted pellet.
Immunocytochemistry Monoclonal
antibodies
The monoclonal anti-ER (H-222) was raised against purified human MCF-7 cell extranuclear ER‘and was provided by Abbott Laboratories (North Chicaeo. IL). The PR monoclonal antibodv fB39) was urovided by Dr. G. Gyeene’(University of Chicago) and’the AR mdnoclonal antibody (ANl-15) was provided by Dr. S. Liao (University of Chicago). A nonspecific control antibody of the same immunoglobulin subclass, raised against Timothy grass pollen antigen B (AT) was provided Dr. A. Malley (ORPRC).
Estrogen
and androgen
receptor assays
We utilized a gradient shift assay, as previously described (13-15) to measure ER and AR in adrenal tissue. The assay involves the incubation of tritiated ligand with fresh tissue at 37 C and the use of specific
monoclonal
antibodies to separate receptor-bound
ligand from free
ligand on sucrose gradients. The advantage of this assay is that low levels of receptors in small samples can be reliably quantified because the specific antibody shifts the labeled receptors away from nonspecifi-
callv labeled molecules and free liaand on the gradient. This facilitates discrimination between specific aid nonspecifically bound ligand and avoids the use of absorbants which may reduce specific binding, thereby leading to an underestimation of receptor concentrations. A series of preliminary studies were conducted to determine the optimal assay conditions for adrenal tissue and these studies are described in Results. Subsequently all assays were performed by the following procedure. Adrenal gland tissue from adult, immature, and neonatal animals was cut into slices of 0.5 mm thickness with a Stadie-Riggs tissue slicer (Arthur H. Thomas Co., Philadelphia, PA). Fetal adrenal tissue was chopped into cubes 0.5-1.0 mm in diameter. The adrenal tissue was rinsed in Trowell’s medium, containing 0.7% (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid), pH 7.0, at 37 C and then incubated in the same medium with the addition of 10 nmol tritiated 17P-estradiol (3H-E2) or tritiated 5a-dihydrotestosterone (NEN DuPont, Wilmington, DE). The incubations were of 60-min duration and were conducted
Slices of -2 mm thickness were cut from adrenal glands in a crosssectional plane extending from the anteromedial apex to the center of the renal depression as described by McNulty et al. (17). The tissue was imbedded in a drop of Tissue-Tek OTC (Miles Laboratories, Naperville, IL) and frozen by plunging in liquid propane (-130 C). The sections were stored over liquid nitrogen until processed further. Cryostat sections (4-6 Frn) were cut at -20 C on a Hacker-Bright cryostat (Hacker Instruments, Fairfield, NJ) and thaw mounted on gelatin-coated glass slides. Tissue sections were prepared for ICC as described by McClellan et aI. (18) using the indirect method with avitin-biotin complex except for the following modifications. Frozen tissue sections were fixed at 4 C in picric acid paraformaldehyde for lo-12 min and then 85% ethanol for 4 min. The slides were rinsed in phosphate-buffered saline (PBS), pH 7.4, placed in sodium borohydride for 4 min, and rinsed again with PBS containing 1% gelatin. Next the tissue sections were incubated with normal rabbit serum for 30 min followed by an incubation with monoclonal anti-ER, PR, AR, or AT. The incubations were conducted at 4 C, in a moist chamber, overnight. The following day the sections were rinsed with PBS and incubated with normal rabbit serum and then with biotinylated anti-rat immunoglobulin G for 30 min at 20 C. The biotin was detected with an avitin-biotin peroxidase kit (Vector Laboratories, Burlingame, CA). All slides that were compared for staining intensity were processed in the same ICC run on the same day and color photomicrographs were taken with planapochromatic lenses on Ektar 25 film.
Statistical
analysis
Biochemical data were analyzed by Student’s t test and, when more than two groups were considered, by one-way analysis of variance followed by Duncan’s multiple range test. Correlation was determined by least squares method.
Results
under an atmosphere of 60% 02, 34% N2, 6% COZ. At the conclusion of the incubations the tissue was frozen and stored over liquid nitrogen prior to further processing. There was no difference in the concentration of ER detected when the tissue was frozen at this stage compared to immediate processing. Frozen adrenal tissue was homogenized in TEDGM buffer (10 mmol Tris, 1.5 mmol ethylenediaminetetraacetic acid [EDTA], 1 mmol dithiotreitol [DDT], 10% vol/vol glycerol, 10 mmol sodium molybdate, pH 7.4) at the rate of 100 mg tissue/ml at 4 C with a glass/glass hand homogenizer. The homogenate was centrifuged at 2,500 X g for 10 min, at 4 C, vielding a crude nuclear pellet, The supernatant was retained as the cytdsolic fraction. The pellet-was washed ihree times with the same volume of TEDGM and the final uellet was resusuended in TEDK buffer (10 mmol Tris, 1.5 mmol EDTAt 1 mmol dithiotreitol, 0.5 mmol KCl, pH 7.4; 200 mg original tissue/l mL) to extract nuclear receptor. Extrac-
tions were for 60 min at 0 C with gentle agitation at 15-min intervals. Following nuclear extraction the suspension was centrifuged at 10,000 x g for 10 min. The pellet was stored at -20 C, prior to assay for DNA (16), and the supernatant was divided into 500 PL aliquots. Each aliquot was added to 2.5 Kg of monoclonal antibody, allowed to incubate for 60 min at 0 C, and then subjected to sedimentation analysis on 5-20% sucrose gradients (prepared in 10 mmol Tris, 1.5 mmol EDTA, 0.5 KCl). Protein solutions (BSA, 4.5s and aldolase, 8S), that were used as sedimentation markers, were layered and run on a parallel gradient. The gradients were centrifuged at 205,000 x g for 18 h at 4 C. Following fractionation of the gradients, each fraction was mixed with Scintiverse BD (Fisher Scientific, Pittsburgh, PA) and radioactivity was quantitated with a model 460-CD Packard liquid scintillation counter. The radioactivity associated with the 8s shifted peak of the gradient profile resulted
from tritiated steroid-receptor
antibody complexes and the quantity of
Steroid
receptor assays
The major assay parameters were investigated in preliminary experiments at least twice. The monoclonal antibodies formed complexes with 3H-E,-labeled receptors and these complexes were detected as peaks, observed in the 8s position, on sucrose gradients (Fig. 1). These peaks were shifted away from free 3H-E2 and nonspecifically bound 3H-E2 at the top of the gradient. As shown in Fig. 1, the 8s peak was absent when a nonspecific antibody of the same immunoglobulin subclass (AT), or when the PR antibody was used in place of the anti-ER under the same conditions with the same tissue. Thus, the labeled ER remained near the top of the gradient and obscured by the presence of free 3H-E2 unless the ER monoclonal antibody was present. The incubation of adrenal tissue slices with *H-E3 at 37 C for increasing time periods resulted in maximal labeling and detection of nuclear ER at 60 min of incubation (Fig. 2). Incubations of 60 min were therefore used for all further studies. There was a slight decrease in ER detection when incubations were extended to 120 min, suggesting that receptor processing and/or degradation may occur at 37 C with longer incubations. Optimal extraction of nuclear ER was obtained with 60 min of extraction at 0 C and with 0.5 mol KC1 as the
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 02 March 2015. at 18:45 For personal use only. No other uses without permission. . All rights reserved.
HIRST ET AL.
310
FIG. 1. Sucrose density gradient profile of 3H-E2 binding in nuclear extracts of adrenal tissue from rhesus monkeys. Slices of fresh tissue were incubated with 10 nmol/L 3H-E, and nuclear extracts of the tissue were mixed with monoclonal antibodies, anti-ER, anti-PR, and with a nonspecific control antibody (antiTimothy grass pollen antigen B, AT) and run on 5-20% sucrose gradient run for 18 h at 205,600 x g. The shaded area indicates the peak resulting from the sedimentation 3H-Ez labeled receptorantibody complexes in the presence of anti-ER. The quantity of ‘H-E2 associated with this peak (shaded area) was used as a measure of ER. No peaks were observed with either anti-PR or AT. The positions of BSA (4.5s) and aldolase (8s) marker proteins are indicated.
- -
Anti-E!?
-.-
Anti-PI?
-
Control
JCE & M. 1992 Vol75.Nol
A%!~e
BSA 4.5s
4
z !r 11 I ‘i
Receptor
Antibody
l,,,,,,,,J,,,,,,,,I
1
4
6
8
10
12
14
16
18
20
I
/,
22
I,
24
Botto:
26 TOD
Fraction
Number
12
14r
1
10
Incubation
30
Time
60
I
