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Receptor progesterone complex in the nuclear fraction of the rat uterus: Demonstration by 3H-progesterone exchange
599
RECEPTOR PROGESTERONE COMPLEX IN THE NUCLEAR FRACTION OF THE RAT UTERUS: DEMONSTRATION BY 'H-PROGESTERONE EXCHANGE
A.J.W. HSUEH, E.J. PECK, JR. AND J.H. CLARK
Department of Cell Biology Baylor College of Medicine Houston, Texas 77025 received
:
915174 ABSTRACT
We have previously shown that 3H-estradiol exchange can be used to measure the quantity of estrogen receptor complex in the nuclear fraction of target tissue cells. This method has been modified for the measurement of the progesterone receptor complex (R,*P) in the nuclear fraction of uterine cells. Nuclear fractions were incubated for 5 hrs. at 15O in the presence of varying concentrations of 3H-progesterone ( sH-P) with or without a 250-fold excess of non-la eled progesterone (P). Rn.P was determined by subtracting th H-P bound in the presence of excess P (non-specific binding) from !? H-P bound in the absence of excess P (total binding). All R,.P studies were done in adult castrate female rats that had received estradiol benzoate (0.4 mg) one week before use. The quantity of Rn*P increased in the uterine nuclear fractions by 280% 30 min. after injection of 5 mg of P. Rn*P was not increased in muscle or fat pad by this treatment. Injections of corticosterone (B), cortisol (F), dexamethasone (Dx) or testosterone (T) failed to increase R,.P. The exchange reaction was specific for P; B, F, Dx or T did not compete. These results demonstrate the existence of a low capacity, high affinity, stereospecific progesterone binding site in the nuclear fraction of the uterus. INTRODUCTION It is generally accepted that steroid hormones are bound by specific receptor molecules that are present in the cytoplasm of target tissue cells (l-6). This receptor steroid complex undergoes
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translocation to nuclear sites where it probably stimulates events that are important to the regulation of metabolic and growth functions (1,5,6). Although the cytoplasmic progesterone receptor has been described in some detail in both avian and mammalian systems (7-14), the nuclear receptor progesterone complex (R,.P) has been studied in detail only in the chick oviduct and little information concerning Rn*P is available in mammals (15,16). We have previously shown that the translocation and accumulation of the estrogen receptor complex in the uterine nuclear fraction could be measured by the 3H-estradiol exchange assay (17). This method is based on the exchange of 3H-estradiol with nonlabeled estrogen bound to the receptor which is present as a receptor estrogen complex in the nucleus.
In theory, this method
should be applicable to the measurement of all steroid hormone nuclear receptor complexes and in this paper we demonstrate that it can be used to characterize the Rn*P in the rat uterus. METHODS AND MATERIALS Ovariectomized adult female rats (body weight 200 g - 250 g) of the Holtzman strain were purchased from Hormone Assay (Chicago, Ill.). The animals were maintained in air conditioned quarters and given food and water -ad lib. One week before sacrifice, all animals were injected with 0.4 mg estradiol-176 3 benzoate (EB) in 0.4 ml vegetable oil. In experiments designed to examine the translocation of the cytoplasmic receptors to the particulate fractions, different steroids were dissolved in 0.25 mldimethylsulfoxide (DMSO) and injected subcutaneously. Control animals were injected with 0.25 ml DMSO. l,2-3H progesterone (New England Nuclear Corp., Boston, Mass., Sp. act. 48 Ci/mM) was examined for purity by t.1.c. Animals were killed by decapitation and the uteri were cleaned and slit. The tissues were weighed with a torsion balance
Tris-HCl with and washed in cold Tris-gl cerol (TG) buffer (lO.rrM_ 10% glycerol (v/v), pH 7.2J . All subsequent steps were performed at 4 unless otherwise indicated. The tissues were homogenized on all-glass Kontes homogenizer with a motor-driven pestle. The nuclear myofibrillar fraction was obtained by centrifugation of the homogenate at 800 g for 10 min. This fraction was washed three times with 3 ml of buffer and each wash was followed by centrifugation at 800 g for 10 min. The washed pellet was suspended in TG buffer to a concentration equivalent to 80 mg tissue/ml and gently rehomogenized. Portions (0.5 ml) of this suspension were dispensed into two series of tubes, A and B, containing 0.2 ml of buffer. Series A contained 75 nM 3H-progesterone and was used to determine the total ount of 3H-progesterone exchanged. Tubes in series B contained YH-progesterone as in series A plus a 250-fold excess of non-radioactive progesterone. The nuclear fractions were incubated with shaking at 15OC for 5 hrs. Following incubation, 3 ml TG buffer was added and the samples were centrifuged at 800 g for 10 min. Pellets were washed three times with TG buffer and extracted with 3 ml ethanol. The ethanol extract was then added to 10 ml Scintillation fluid (99.5% Toluene; 0.45% 2,5-diphenyloxazole; 0.5% 1,4-bis-(5-phenyl-oxazol-2-yl) benzene). The concentration of 3H-progesterone that was specifically bound was determined by subtracting results obtained in series B from those of series A. Since all animals received identical pretreatment with EB and subsequent hormonal treatment did not alter the DNA, protein or wet weight of the uterus, we have expressed the amount of specifically bound 3H-progesterone as dpm/mg of wet weight. Radioactivity was determined with a Packard Liquid Scintillation Spectrometer at 25% efficiency. RESULTS AND DISCUSSION A.
The determination of optimal time and temperature for 3Hprogesterone exchange. The 3H-estradiol exchange assay for the determination of re-
ceptor estrogen complex in uterine nuclei was done at 37'C for 30 min. and a stochiometric relationship between cytoplasmic receptors and nuclear receptor estrogen complexes was demonstrated (17). Early attempts to use these assay conditions for the measurement of the nuclear receptor progesterone complex (Rn*P) failed. The
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possibility that the rate of degradation of R,*P during the assay was great with respect to rate of exchange, such that little R,.P could be measured, was examined. Ovariectomized rats that were treated with estradiol benzoate (EBX) were injected with 5 mg progesterone 15 minutes before sacrifice and the uterine nuclear fraction was prepared as described in Methods and Materials section. Uterine nuclear fractions were subsequently incubated at 15O, 25O, and 37OC for various intervals from 30 minutes to 24 hours and the specifically bound 3H-progesterone was determined. The quantity of bound 3H-progesterone at 25O and 37OC was significant, but the Rn*P appears to undergo rapid degradation at these temperatures, thus making the assay difficult to use (Fig. 1).
The optimal temperature
for the assay appears to be 15'C (Fig. 1).
0
4
8
12 TIME
16 (hours
20
24
28
1
Figure 1. The effect of incubation tim and temperature on the 3Hprogesterone exchange assay. Specific 5H-progesterone binding was determined as described in the text and the results were expressed as dpm/mg wet weight. Each value represents the mean + S.E.M. for 3- 4 determinations.
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These results indicate that at reduced temperatures the rate of exchange of 3H-progesterone for non-labeled progesterone is probably greater than the rate of degradation of the receptor. The observation that degradation or inactivation of the progesterone receptor is taking place during the incubation period of the assay has important implications (Fig.
1).
Obviously if degradation is going
on at the same time as 3H-progesterone exchange then this assay does not measure all receptor sites. Hence the method as presented is only semiquantitative. B.
Identificationof a high affinity, low capacity progesterone binding site in the nuclear fractions Uterine tissues were taken from EBX rats 30 minutes after an
injection of 5 mg of progesterone. The nuclear fractions were prepared and incubated at various concentrations of 3H-progesterone (6, 15, 30 and 75 nM total binding - group A, Fig. 2) or the same concentration of 3H-progesterone plus 250-fold excess of non-radioactive progesterone (non-specific binding - group B, Fig. 2) as described in Methods and Materials section. The difference between group A and B represents the quantity of 'H-progesterone that was specifically bound. Analysis of these results by a double reciprocal plot gives an apparent dissociation constant, 4, 1O"8 &
of about
These results indicate that following an injection of pro-
gesterone there are low capacity, high affinity progesterone binding sites present in the uterine nuclear fraction.
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Figure 2. The demonstration by 3H-progesterone exchange of a high affinity, low capacity progesterone binding site in rat uterine nucTei. Uterin nuclear pellets were incubated with various concentrations of Ti H-progesterone A (total binding) or SH-progesterone plus a 250-fold excess of non-labeled progesterone 6 (non-specific binding), The non-specific binding, B, was subtracted from the total binding, A, to yield the specific binding, C. C.
Stereospecificity of receptor progesterone complex Because the nuclear binding sites involved in the 3H-proges-
terone exchange are expected to exhibit a high specificity for progestational compounds, the effect of various hormones on the exchange of 3H-progesterone in uterine nuclear fractions was tested. Uterine nuclear fractions were prepared from EBX rats 15 minutes after an injection of 5 mg of progesterone. Non-radioactive hormones at lOO-fold the concentration of SH-progesterone were added
to the assay tubes and the exchange assay performed as described fn Materials and Methods. As shown above and in Table 1, progesterone acts as a competitive inhibitor for 3H-progesterone exchange, whereas corticosterone, cortisol, dexamethasone (9-fluor-118, 17, Zltrihydroxy-16u-methyl-1,4-pregnandiene-3,20-dione) and testosterone had no competitive effect (Table 1). These results demonstrate that binding to the nuclear receptor progesterone complex is a stereospecific process. In addition, the stereospecificityof this binding site excludes the possibility of contamination by transcortin-like molecules as has been observed in the uterine cytosol (8,12,13). Nonradioactive Hormone Added
3H-Progesterone Bound (Total, dpm/mg)
3H-Progesterone Bound (Specific, dpm/mg)
None
4440 + 160
-_
Progesterone
2720 + 286
1720
Corticosterone
4200 + 230
N.S.*
Cortisol
4350 2 120
N.S.
Testosterone
4430 +I 152
N.S.
Dexamethasone
4330 + 133
N.S.
*
No signiffcant specific 3H-progesterone binding
Table 1. Stereospecificityof the nuclear receptor progesterone complex. Each compound was added to the assay buffer at 100 x the concentration of 3H-progesterone and incubated with uterfne pellets for 5 hr., 15OC.
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The influence of various steroids on the translocation of receptor progesterone complex into the nucleus in vivo
If the accumulation of Rn.P is the result of translocation of cytoplasmic progesterone receptors, then this accumulation should be specific for progestational other steroid hormones. terone, corticosterone, 0.25 ml DMSO.
compounds and not be affected by
Rats were injected with 5 mg of progescortisol, dexamethasone,
or testosterone
in
Thirty minutes after injection, the uterine nuclear
fraction was prepared and used in the incubation procedure described above (Table 2).
It can be seen that the injection of progesterone
results in a substantial control value.
increase of specific binding sites over
In contrast, an injection of cortisol, corticoster-
one, dexamethasone,
or testosterone failed to show any significant
effect on the number of receptors in the nucleus of the rat uterus. These data demonstrate the specific nature of the progesterone induced accumulation of the receptor by the uterine nuclear fraction. The possible origin of the low, but significant quantity of R,.,*P observed in the rats that received only DMSO was examined. were ovariectomized
and adrenalectomized
Animals
and 4 days later they were
injected with 0.4 mg of estradiol benzoate.
Seven days after in-
jection they were killed and the amount of Rn*P was examined following an injection of MSO.
As shown in Table 2, the level of
Rn*P in these animals is decreased and this suggests that the quantity of R,.P that was observed in the non-adrenalectomized was the result of adrenal progesterone.
animals
Compound Injected
Specific 3H-progesterone Bound (dpm/mg)
DMSO
853 + 131
DMSO in adrenalectomized and ovariectomited rats
442 + 190
Progesterone
2100 5 121
Corticosterone
712 + 134
Cortisol
837 +168
Dexamethasone
535 + 168
Testosterone
558 + 173
Table 2. The effects of various hormones on the quantity of nuclear receptor progesterone complex following injection. Animals were injected with the various compounds as described in the text. Thirty minutes later the 3H-progesteroneexchange assay was performed. E.
Influence of progesterone on the number of nuclear bindinq sites in target and non-tarqet tissues
In order to test the tissue specificity of the accumulation of R,*P following an injection of progesterone, EBX rats were injected with 5 mg progesterone in 0.25 ml DMSO or 0.25 ml DMSO alone. Fifteen minutes later the concentrations of R,.P was determined in the uterine tissue, abdominal muscle and fat pads (Table 3).
Pro-
gesterone treatment results in a marked increase in specific nuclear binding sites in the uterus. However, significant levels of R,,.P were not observed in muscle or fat pad nuclear fractions following progesterone treatment. These data suggest a tissue specific accumulation of Rn*P that is induced by progesterone.
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3H-progesterone DMSO
Tissue
Uterus
853 t 131
Muscle
414 2
85
325 + 76
45 2
23
N.D.*
Fat Pad
*
Bound (dpm/mq) Progesterone
1744 2 67
Not detectable
Figure 3. The effect of progesterone treatment on the quantity of nuclear receptor progesterone complex in target and non-target tissues. Animals were injected with 5 mg of progesterone and the amount of Rn*P was determined 15 minutes later.
0.0 0
I
I
I
I
15
30
60
90
TIME
tminutesJ
Figure 3. The time course of the accumulation and retention of the receptor-progesterone complex by rat uterine nuclei. Animals were injected with 5 mg of progesterone and the amount of specific 3Hprogesterone bound was determined at the times indicated.
s F.
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Time course of the nuclear accumulation and retention of R,*P To study the effect of a single dose of progesterone on the
concentration of Rn.P, EBX rats were injected with 5 mg progesterone in 0.25 ml MO.
At various intervals after injection, the 3H-pro-
gesterone exchange assay was performed as described above. The results shown in Figure 3 ind_icatethat an injection of progesterone results in the rapid accumulation of R,.P by the uterine nuclear fraction. This is followed by a gradual decline in the number of sites between 30 and 90 minutes (Fig. 3).
This accumulation and
retention of R,.P by the nucleus probably results from the translocation of the cytoplasmic receptor progesterone complex to acceptor sites in the nucleus as has been proposed for the progesterone receptor in the chick oviduct (18)
and for the estrogen receptor in
the rat uterus (19). CONCLUSION This report demonstrates that the principles and methodologies of the 3H-estradiol exchange assay can be used in a 3H-progesterone exchange system to demonstrate the presence of a receptor progesterone complex in the nuclei of uterine cells. This method enables one to evaluate the basic criteria for steroid hormone receptors, i.e., finite binding capacity, high affinty and stereospecificity and overcomes the complexities of studying cytosol preparations that may be contaminated with transcortin. The method should be useful for the evaluation of non-labeled progestational and anti-
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progestational compounds as we have done for the analysis of the mechanism of action of the anti-estrogen, nafoxidine (Upjohn, 11,100 A,ZO). Further improvement of this technique will not only provide a quantitative method for the estimation of Rn*P concentration but will also permit the study of the relationships that exist between R,*P and progesterone induced responses in viva. ACKNOWLEDGEMENTS This work was supported by grants from The National Institutes of Health (HD 04985), and The American Cancer Society (BC-92). We thank L. Hsueh for her excellent technical assistance. REFERENCES
1.
Gorski, J., Toft, D Shyamala, G., Smith, D., and Noties, A., RECENT PROGR. HORM.'liES.24, 45 (1968).
2.
Fang, S., Anderson, K.M., and Liao,S., J. BIOL. CHEM. 244, 6584 (1969).
3.
Baxter, J.D., and Tomkins, G.M., PROC. NAT. ACAD. SCI. WASH. 65, 709 (1970).
4.
Swaneck, G.E., Chu, L.L.H., and Edelman, I.S., J. BIOL. CHEM. 245, 5382 (1970).
5.
Jensen, E.V., and DeSombre, E.R., ANN. REV. BIOCHEM. 41, 203 (1972).
6.
O'Malley, B.W., and Means, A.R., SCIENCE 183, 610 (1974).
7.
Sherman, M-R., Corvol, P.L., and O'Malley, B.W., J. BIOL. CHEM. 245, 6085 (1970).
8.
Milgrom, E., and Baulieu, E.E., ENDOCRINOLOGY 87, 276 (1970).
9.
McGuire, J.L.,
10.
Reel, J.R., Van Dewark, S.D., Shih, Y., and Callantine, M.R., STEROIDS 18, 441 (1971).
and DeDella, C., ENDOCRINOLOGY 88, 1099 (1971).
11.
Wiest, W.G., and Rao, B.P., ADVANCES IN THE BIOSCIENCES,Vol. 7. "Schering Workshop on Steroid Hornme Receptors," Pergamon Press, Vieneg, New York, 1971, p. 251.
12.
Feil, P.D., Glasser, S.R., Toft, D.O., and O'Malley, B.W., ENDOCRINOLOGY 91, 738 (1972).
13.
Faber, L.E., Sandmann, M.L., and Stavely, H.E., ENDOCRINOLOGY 93, 74 (1973).
14.
Kontula, K., Janne, O., Janne, J., and Vihko, R., BIOCHEM. BIOPHYS. RES. COt&lUN.47, 596 (1972).
15.
O’Malley,
16.
Rao, B.R., Wiest, W.G., and Allen, W.M., ENDOCRINOLOGY 92, 1129 (1973).
17.
Anderson, J.N., Clark, J.H., and Peck, E.J., 126, 561 (1972).
18.
Spelsberg, T.C., Steg les, CHEM. 246, 4188 (1971 3 .
19.
Clark, J.H., Anderson, J.N., dnd Peck, E.J., Jr., HORMONE RECEPTORS AND REPRODUCTIVE HORMONES, Adv. Exptl. Med. and Biol., B.W. O'Malley and A.R. Means (eds. ) 1972, p. 15.
20.
B.W., and Toft, D.O., J. BIOL. CHEM. 246, 1117 (1971).
Clark, J.H., 707 (1973).
Anderson, J.N.,
Jr.
BIOCHEM.J.
A.W., and O'Malley, B.W., J. BIOL.
and Peck, E.J., Jr.,
STEROIDS22,
RECEPTOR PROGESTERONE COMPLEX IN THE NUCLEAR FRACTION OF THE RAT UTERUS: DEMONSTRATION BY 'H-PROGESTERONE EXCHANGE
A.J.W. HSUEH, E.J. PECK, JR. AND J.H. CLARK
Department of Cell Biology Baylor College of Medicine Houston, Texas 77025 received
:
915174 ABSTRACT
We have previously shown that 3H-estradiol exchange can be used to measure the quantity of estrogen receptor complex in the nuclear fraction of target tissue cells. This method has been modified for the measurement of the progesterone receptor complex (R,*P) in the nuclear fraction of uterine cells. Nuclear fractions were incubated for 5 hrs. at 15O in the presence of varying concentrations of 3H-progesterone ( sH-P) with or without a 250-fold excess of non-la eled progesterone (P). Rn.P was determined by subtracting th H-P bound in the presence of excess P (non-specific binding) from !? H-P bound in the absence of excess P (total binding). All R,.P studies were done in adult castrate female rats that had received estradiol benzoate (0.4 mg) one week before use. The quantity of Rn*P increased in the uterine nuclear fractions by 280% 30 min. after injection of 5 mg of P. Rn*P was not increased in muscle or fat pad by this treatment. Injections of corticosterone (B), cortisol (F), dexamethasone (Dx) or testosterone (T) failed to increase R,.P. The exchange reaction was specific for P; B, F, Dx or T did not compete. These results demonstrate the existence of a low capacity, high affinity, stereospecific progesterone binding site in the nuclear fraction of the uterus. INTRODUCTION It is generally accepted that steroid hormones are bound by specific receptor molecules that are present in the cytoplasm of target tissue cells (l-6). This receptor steroid complex undergoes
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translocation to nuclear sites where it probably stimulates events that are important to the regulation of metabolic and growth functions (1,5,6). Although the cytoplasmic progesterone receptor has been described in some detail in both avian and mammalian systems (7-14), the nuclear receptor progesterone complex (R,.P) has been studied in detail only in the chick oviduct and little information concerning Rn*P is available in mammals (15,16). We have previously shown that the translocation and accumulation of the estrogen receptor complex in the uterine nuclear fraction could be measured by the 3H-estradiol exchange assay (17). This method is based on the exchange of 3H-estradiol with nonlabeled estrogen bound to the receptor which is present as a receptor estrogen complex in the nucleus.
In theory, this method
should be applicable to the measurement of all steroid hormone nuclear receptor complexes and in this paper we demonstrate that it can be used to characterize the Rn*P in the rat uterus. METHODS AND MATERIALS Ovariectomized adult female rats (body weight 200 g - 250 g) of the Holtzman strain were purchased from Hormone Assay (Chicago, Ill.). The animals were maintained in air conditioned quarters and given food and water -ad lib. One week before sacrifice, all animals were injected with 0.4 mg estradiol-176 3 benzoate (EB) in 0.4 ml vegetable oil. In experiments designed to examine the translocation of the cytoplasmic receptors to the particulate fractions, different steroids were dissolved in 0.25 mldimethylsulfoxide (DMSO) and injected subcutaneously. Control animals were injected with 0.25 ml DMSO. l,2-3H progesterone (New England Nuclear Corp., Boston, Mass., Sp. act. 48 Ci/mM) was examined for purity by t.1.c. Animals were killed by decapitation and the uteri were cleaned and slit. The tissues were weighed with a torsion balance
Tris-HCl with and washed in cold Tris-gl cerol (TG) buffer (lO.rrM_ 10% glycerol (v/v), pH 7.2J . All subsequent steps were performed at 4 unless otherwise indicated. The tissues were homogenized on all-glass Kontes homogenizer with a motor-driven pestle. The nuclear myofibrillar fraction was obtained by centrifugation of the homogenate at 800 g for 10 min. This fraction was washed three times with 3 ml of buffer and each wash was followed by centrifugation at 800 g for 10 min. The washed pellet was suspended in TG buffer to a concentration equivalent to 80 mg tissue/ml and gently rehomogenized. Portions (0.5 ml) of this suspension were dispensed into two series of tubes, A and B, containing 0.2 ml of buffer. Series A contained 75 nM 3H-progesterone and was used to determine the total ount of 3H-progesterone exchanged. Tubes in series B contained YH-progesterone as in series A plus a 250-fold excess of non-radioactive progesterone. The nuclear fractions were incubated with shaking at 15OC for 5 hrs. Following incubation, 3 ml TG buffer was added and the samples were centrifuged at 800 g for 10 min. Pellets were washed three times with TG buffer and extracted with 3 ml ethanol. The ethanol extract was then added to 10 ml Scintillation fluid (99.5% Toluene; 0.45% 2,5-diphenyloxazole; 0.5% 1,4-bis-(5-phenyl-oxazol-2-yl) benzene). The concentration of 3H-progesterone that was specifically bound was determined by subtracting results obtained in series B from those of series A. Since all animals received identical pretreatment with EB and subsequent hormonal treatment did not alter the DNA, protein or wet weight of the uterus, we have expressed the amount of specifically bound 3H-progesterone as dpm/mg of wet weight. Radioactivity was determined with a Packard Liquid Scintillation Spectrometer at 25% efficiency. RESULTS AND DISCUSSION A.
The determination of optimal time and temperature for 3Hprogesterone exchange. The 3H-estradiol exchange assay for the determination of re-
ceptor estrogen complex in uterine nuclei was done at 37'C for 30 min. and a stochiometric relationship between cytoplasmic receptors and nuclear receptor estrogen complexes was demonstrated (17). Early attempts to use these assay conditions for the measurement of the nuclear receptor progesterone complex (Rn*P) failed. The
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possibility that the rate of degradation of R,*P during the assay was great with respect to rate of exchange, such that little R,.P could be measured, was examined. Ovariectomized rats that were treated with estradiol benzoate (EBX) were injected with 5 mg progesterone 15 minutes before sacrifice and the uterine nuclear fraction was prepared as described in Methods and Materials section. Uterine nuclear fractions were subsequently incubated at 15O, 25O, and 37OC for various intervals from 30 minutes to 24 hours and the specifically bound 3H-progesterone was determined. The quantity of bound 3H-progesterone at 25O and 37OC was significant, but the Rn*P appears to undergo rapid degradation at these temperatures, thus making the assay difficult to use (Fig. 1).
The optimal temperature
for the assay appears to be 15'C (Fig. 1).
0
4
8
12 TIME
16 (hours
20
24
28
1
Figure 1. The effect of incubation tim and temperature on the 3Hprogesterone exchange assay. Specific 5H-progesterone binding was determined as described in the text and the results were expressed as dpm/mg wet weight. Each value represents the mean + S.E.M. for 3- 4 determinations.
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603
These results indicate that at reduced temperatures the rate of exchange of 3H-progesterone for non-labeled progesterone is probably greater than the rate of degradation of the receptor. The observation that degradation or inactivation of the progesterone receptor is taking place during the incubation period of the assay has important implications (Fig.
1).
Obviously if degradation is going
on at the same time as 3H-progesterone exchange then this assay does not measure all receptor sites. Hence the method as presented is only semiquantitative. B.
Identificationof a high affinity, low capacity progesterone binding site in the nuclear fractions Uterine tissues were taken from EBX rats 30 minutes after an
injection of 5 mg of progesterone. The nuclear fractions were prepared and incubated at various concentrations of 3H-progesterone (6, 15, 30 and 75 nM total binding - group A, Fig. 2) or the same concentration of 3H-progesterone plus 250-fold excess of non-radioactive progesterone (non-specific binding - group B, Fig. 2) as described in Methods and Materials section. The difference between group A and B represents the quantity of 'H-progesterone that was specifically bound. Analysis of these results by a double reciprocal plot gives an apparent dissociation constant, 4, 1O"8 &
of about
These results indicate that following an injection of pro-
gesterone there are low capacity, high affinity progesterone binding sites present in the uterine nuclear fraction.
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Figure 2. The demonstration by 3H-progesterone exchange of a high affinity, low capacity progesterone binding site in rat uterine nucTei. Uterin nuclear pellets were incubated with various concentrations of Ti H-progesterone A (total binding) or SH-progesterone plus a 250-fold excess of non-labeled progesterone 6 (non-specific binding), The non-specific binding, B, was subtracted from the total binding, A, to yield the specific binding, C. C.
Stereospecificity of receptor progesterone complex Because the nuclear binding sites involved in the 3H-proges-
terone exchange are expected to exhibit a high specificity for progestational compounds, the effect of various hormones on the exchange of 3H-progesterone in uterine nuclear fractions was tested. Uterine nuclear fractions were prepared from EBX rats 15 minutes after an injection of 5 mg of progesterone. Non-radioactive hormones at lOO-fold the concentration of SH-progesterone were added
to the assay tubes and the exchange assay performed as described fn Materials and Methods. As shown above and in Table 1, progesterone acts as a competitive inhibitor for 3H-progesterone exchange, whereas corticosterone, cortisol, dexamethasone (9-fluor-118, 17, Zltrihydroxy-16u-methyl-1,4-pregnandiene-3,20-dione) and testosterone had no competitive effect (Table 1). These results demonstrate that binding to the nuclear receptor progesterone complex is a stereospecific process. In addition, the stereospecificityof this binding site excludes the possibility of contamination by transcortin-like molecules as has been observed in the uterine cytosol (8,12,13). Nonradioactive Hormone Added
3H-Progesterone Bound (Total, dpm/mg)
3H-Progesterone Bound (Specific, dpm/mg)
None
4440 + 160
-_
Progesterone
2720 + 286
1720
Corticosterone
4200 + 230
N.S.*
Cortisol
4350 2 120
N.S.
Testosterone
4430 +I 152
N.S.
Dexamethasone
4330 + 133
N.S.
*
No signiffcant specific 3H-progesterone binding
Table 1. Stereospecificityof the nuclear receptor progesterone complex. Each compound was added to the assay buffer at 100 x the concentration of 3H-progesterone and incubated with uterfne pellets for 5 hr., 15OC.
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The influence of various steroids on the translocation of receptor progesterone complex into the nucleus in vivo
If the accumulation of Rn.P is the result of translocation of cytoplasmic progesterone receptors, then this accumulation should be specific for progestational other steroid hormones. terone, corticosterone, 0.25 ml DMSO.
compounds and not be affected by
Rats were injected with 5 mg of progescortisol, dexamethasone,
or testosterone
in
Thirty minutes after injection, the uterine nuclear
fraction was prepared and used in the incubation procedure described above (Table 2).
It can be seen that the injection of progesterone
results in a substantial control value.
increase of specific binding sites over
In contrast, an injection of cortisol, corticoster-
one, dexamethasone,
or testosterone failed to show any significant
effect on the number of receptors in the nucleus of the rat uterus. These data demonstrate the specific nature of the progesterone induced accumulation of the receptor by the uterine nuclear fraction. The possible origin of the low, but significant quantity of R,.,*P observed in the rats that received only DMSO was examined. were ovariectomized
and adrenalectomized
Animals
and 4 days later they were
injected with 0.4 mg of estradiol benzoate.
Seven days after in-
jection they were killed and the amount of Rn*P was examined following an injection of MSO.
As shown in Table 2, the level of
Rn*P in these animals is decreased and this suggests that the quantity of R,.P that was observed in the non-adrenalectomized was the result of adrenal progesterone.
animals
Compound Injected
Specific 3H-progesterone Bound (dpm/mg)
DMSO
853 + 131
DMSO in adrenalectomized and ovariectomited rats
442 + 190
Progesterone
2100 5 121
Corticosterone
712 + 134
Cortisol
837 +168
Dexamethasone
535 + 168
Testosterone
558 + 173
Table 2. The effects of various hormones on the quantity of nuclear receptor progesterone complex following injection. Animals were injected with the various compounds as described in the text. Thirty minutes later the 3H-progesteroneexchange assay was performed. E.
Influence of progesterone on the number of nuclear bindinq sites in target and non-tarqet tissues
In order to test the tissue specificity of the accumulation of R,*P following an injection of progesterone, EBX rats were injected with 5 mg progesterone in 0.25 ml DMSO or 0.25 ml DMSO alone. Fifteen minutes later the concentrations of R,.P was determined in the uterine tissue, abdominal muscle and fat pads (Table 3).
Pro-
gesterone treatment results in a marked increase in specific nuclear binding sites in the uterus. However, significant levels of R,,.P were not observed in muscle or fat pad nuclear fractions following progesterone treatment. These data suggest a tissue specific accumulation of Rn*P that is induced by progesterone.
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3H-progesterone DMSO
Tissue
Uterus
853 t 131
Muscle
414 2
85
325 + 76
45 2
23
N.D.*
Fat Pad
*
Bound (dpm/mq) Progesterone
1744 2 67
Not detectable
Figure 3. The effect of progesterone treatment on the quantity of nuclear receptor progesterone complex in target and non-target tissues. Animals were injected with 5 mg of progesterone and the amount of Rn*P was determined 15 minutes later.
0.0 0
I
I
I
I
15
30
60
90
TIME
tminutesJ
Figure 3. The time course of the accumulation and retention of the receptor-progesterone complex by rat uterine nuclei. Animals were injected with 5 mg of progesterone and the amount of specific 3Hprogesterone bound was determined at the times indicated.
s F.
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W-tOXDI
Time course of the nuclear accumulation and retention of R,*P To study the effect of a single dose of progesterone on the
concentration of Rn.P, EBX rats were injected with 5 mg progesterone in 0.25 ml MO.
At various intervals after injection, the 3H-pro-
gesterone exchange assay was performed as described above. The results shown in Figure 3 ind_icatethat an injection of progesterone results in the rapid accumulation of R,.P by the uterine nuclear fraction. This is followed by a gradual decline in the number of sites between 30 and 90 minutes (Fig. 3).
This accumulation and
retention of R,.P by the nucleus probably results from the translocation of the cytoplasmic receptor progesterone complex to acceptor sites in the nucleus as has been proposed for the progesterone receptor in the chick oviduct (18)
and for the estrogen receptor in
the rat uterus (19). CONCLUSION This report demonstrates that the principles and methodologies of the 3H-estradiol exchange assay can be used in a 3H-progesterone exchange system to demonstrate the presence of a receptor progesterone complex in the nuclei of uterine cells. This method enables one to evaluate the basic criteria for steroid hormone receptors, i.e., finite binding capacity, high affinty and stereospecificity and overcomes the complexities of studying cytosol preparations that may be contaminated with transcortin. The method should be useful for the evaluation of non-labeled progestational and anti-
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progestational compounds as we have done for the analysis of the mechanism of action of the anti-estrogen, nafoxidine (Upjohn, 11,100 A,ZO). Further improvement of this technique will not only provide a quantitative method for the estimation of Rn*P concentration but will also permit the study of the relationships that exist between R,*P and progesterone induced responses in viva. ACKNOWLEDGEMENTS This work was supported by grants from The National Institutes of Health (HD 04985), and The American Cancer Society (BC-92). We thank L. Hsueh for her excellent technical assistance. REFERENCES
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