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Oestrogen receptor components of human endometrium
Jowmrl of Swoid Biochrmisrr,r Vol. 15. pp. 313 to 316. 1981 Prmted m Great Britain. All rights reserved
0022-4731/81/010313-04M2.00m CopyrIght Q 1981 Pergamon Press Ltd
OESTROGEN RECEPTOR COMPONENTS HUMAN ENDOMETRIUM
OF
HANNELORRBRA~JNSBRRGand JULIE E. FTLAEGER Department of Chemical Pathology, St. Mary’s Hospital Medical School, London W2 lPG, England SUMMARY Normal human endometrial tissues contain two distinguishable types of low capacity oestrogen receptor sites. Both types are stable at 4 and 15°C but one is highly labile at 30°C and above. In cytosol preparations both types are stable at 22°C at which temperature oestradiol dissociates from receptor
complexes. In nuclear preparations the more labile form is lost rapidly during exchange at 22°C. The presence of KC1 in nuclear homogenates reduced binding of oestradiol to both forms of receptor.
INTRODUCTION
During an investigation of techniques for receptor analysis it was noted that the low-capacity oestrogen binding sites of normal human endometrial tissue included two distinguishable moieties. The present investigation was undertaken to demonstrate the existence of two species of receptor in cytosol and nuclear preparations, and to examine their relative stability under conditions necessary to effect exchange of endogenous bound steroid. MATERIALS
AND
METHODS
The methods used for tissue fractionation and receptor analysis have been published [l, 23 and are outlined in Figs 1 and 2. Cytosol preparations corresponding to 100 mg tissue per ml buffer were used for experiments with [2,4,6,7-3H}-oestradiol ([‘HI-E,, SA 93 Ci/mmol); further S-fold dilutions were made when 16z-[12’I]-oestradiol ([12sI]-E2, initial SA > lOOOCi/mmol) was used. Washed nuclear pellets [I] were taken up in volumes of buffer corresponding to 400 mg and 100 mg tissue per ml in experiments with C3H]-E2 and [‘251]-E2, respectively. RESULTS
On warming at 40°C. two species of low capacity oestradiol binding sites can be distinguished in preparations from human endometrium (Fig. 3): a labile form which is destroyed within 20 or 30min and a more stable form which is lost more slowly at this temperature. In most samples examined so far the heat-stable form has been present in nuclear homogenates but its proportion has tended to be low in cytosol preparations. Most nuclear preparations contain both forms, but the heat-labile form may be absent in some preparations. Since [3H]-E2 and [1251]-E2 gave similar results, the latter has been used in the present work because of its higher specific activity and the greater sensitivity achievable.
High ionic strength solutions extract nuclear oestradiol receptors incompletely [3], and may preferentially extract one of the two forms of receptor. Figure 4 shows the results of an experiment to examine this possibility. Addition of KCl (final concentration 0.4M) to a nuclear homogenate markedly reduced binding to both receptor moieties. Extraction of intact nuclei with KC1 was incomplete and there appeared to be a loss of both receptors forms. The two types of receptor bound [i2’I]-E2 at simiiarrates (Fig. 5). After 22 h the amounts bound at 4 and at 15°C were similar and there was no evidence of any appreciable loss at these temperatures. At 30°C approximately 5% of the stable form and 16% of the labile form was lost during the first hour of incubation (data not shown). Exchange of bound steroid was therefore studied at lower temperatures. Aliquots (25 ~1) of nuclear homogenate were equilibrated with [ ‘251]-E2 in buffer (25 ~1, final concentration 47 PM) at 4°C for 17 h. A lOO-fold excess of radioinert E2 in 5 ~1 buffer was added to one series of tubes and 5 4 buffer only to another (controls). Dissociation of radiolabelled steroid was followed for a period of 27 h. At 4’C there was no loss of radiolabelled steroid from either type of receptor in the controls and in the presence of radioinert E, (data not shown). Thus both types of binding sites are stable and do not dissociate from receptor at 4°C. Figure 6 shows the results of a similar experiment at 22°C. The cytosol preparation contained only a trace of the more stable receptor species. Both forms appeared to be stable at 22°C and exchange was complete after 22 h. When the nuclear homogenate was used, there was little or no loss of the more stable form of receptor. but binding to the labile form in the controls had decreased appreciably by 22 h. Exchange assays at this temperature would, therefore, lead to losses of the labile form of receptor. The rate of dissociation of Et from calf uterine receptors increases in the presence of sodium thiocyanate [4]. A concentration of 0.5 M sodium thiocyanate caused incomplete precipitation of receptors by 313
HANNELORE BRAUNSBERG and JULIEE. PFLAEGER
314
PREPARATION OF TISSUE FRACTIONS.
RECEPTOR ASSAY
@=-T-ml
cl A
Fig. 1. Preparation of tissue fractions. polyethylene glycol, and there was no evidence of dissociation from human endometrial cytopfasmic or nuclear receptors at 4°C. DISCUSSION
The results presented could perhaps be explained without postulating the existence of two distinct
itif%&?~.
Fig. 2. Assay of heat-labile and heat-stable receptor species. Twenty-five ~1 of cytosol or nuclear preparations were incubated with 25 ~1 buffer containing C3H]-E, or [t2sI]-Et. Non-saturable binding was estimated in the presence of a 200-fold excess of diethylstilboestrol (DES).
receptor types. The decrease in binding of radiolabelled steroid after warming (Fig. 3) might be due to dilution by endogenous steroid released from binding sites. This is unlikely since the magnitude of change
. . CYTOSOL 2n-E2.
\
20 5. cYlosoL
,251~22.
h
Fig. 3. Etfect of warming on receptor binding in cytosol and nuclear preparations. After warming at 40°C for different times, samples were equilibrated at 4°C for 24 h with (a) [‘HI-E,, 1.3 nM. (b) [‘HI-E,, 730pM. (c) [‘2S1]-E,. 93 pM. (d) [*z51]-E2, 45 pM: labelled E, only (0). labelled E2 + DES (0).
Components of endometrial oestrogen receptors
l’r
.
I
.
+DES. 0
4
lo
30
20
40
4. b MJaEM
I,
:
nowGEN4l.E
+ UCI.
0 0
s
-DES.
+oEs. 0
x)
20
1 40
30
4 c. ICI
EXTRACT.
3 2 1 ,b= 01
-0Es. o
___;f
l
+m. 0
20
lo TIME
HEATED.
30 hnhutms)
40
Fig. 4. E&t of KCI (0.4 M) on receptor binding by nuclear homogenate and extraction of whole nuclei with 0.4M KCI. A nuclear suspension was divided into two parts. One part was homogenized and samples were warmed at 40°C for different times after addition of buffer only (a) or of buffer containing KCf (b). Intact nuclei were extracted with KC1 at 4” for 30 min and samples of extract warmed for different times (c). All samples were finally equilibrated with [ rz51]-E1 (1OOpM) at 4°C for 22 h.
would require endogenous levels greater than those reported [S, 63. Another possibility is that the labile and stable forms might represent unbound and bound receptor sites, respectively. The results in Fig. 5 and the fact that no exchange takes place at 4°C make this unlikely. The stable form could result from transformation (activation?) of the labile form. This would only be consistent with results in Figs 3 and 4 if the product contained fewer binding sites than the precursor, a possibility which cannot be excluded. The two forms could represent cytoplasmic (labile and nuclear (stable) receptor species, the presence of both in cytosol or nuclear preparations being due to contamination of one with the other. This possibility is under investigation. but the high proportions of the labile form in some nuclear preparations make it unlikely. That two distinguishable low capacity receptor types may be present in nuclear preparations is further supported by the differences in stability of steroid-bound sites observed at 22°C (Fig. 6) and the difference in loss at 3O’C. The evidence thus supports the existence of two
315
oestrogen receptor forms present in varying proportions in cytosol and nuclear preparations. Methods which only use “saturability” to distinguish receptor sites from “non-specific” binding estimate the sum of the two types. Saturation analysis (data not shown) indicates that both the heat-labile and the heat stable forms have high affinity (Kd of the order of lo-” to lo-i” M) and low capacity. Neither species thus corresponds to Type II receptors [33. The results shown in Fig. 4 demonstrate a loss of nuclear oestrogen receptor binding sites in the presence of KCl. It is possible that similar losses are incurred when KC1 extraction is used to estimate nuclear receptors. Our results differ in several respects from those of Bayard et aI.[7j who also studied human endometrial tissues. In their experiments, cytoplasmic oestrogen receptors appeared to be stable at 30°C whereas we find one form to be highly labile. They also found nuclear receptors to be stable during exchange experiments at 30°C using whole nuclei while our nuclear homogenates contained a form which is highly labile at that temperature. These discrepancies may be due to differences in techniques or in the distribution of receptor species present in the tissues examined. The existence of two distinguishable types of high aflinity binding sites in human endometrial nuclei raises the question as to whether they serve different 15
/I=’
f_________8--9--o
.
lo
:
/
5 /
NDN-3FECIFC
t’
or
*
1
’
2
mNDwG.
*
’
3
4
*’
HEAT-LABILE
I
2
3
4
23
0 .
#I’
t
Y’
22
o/__________”
20
’
21
24
l 0
RECEPTOR.
”
21
22
23
24
TIME. (hours)
Fig. 5. Binding of heat-labile and heat-stable nuclear receptors with [ ‘251]-E2 at 4°C (0) and 15” (0).
316
HANNELORE BRAUNSBERG and JULIE E. PFLAEGER CYTOSOL. ,W” E2 *a-
NUCLEI.
b.
a.
M-
.
-
.
lo
C.
_..__z_________--. .
15
.
D
.t II 0
3
‘. ‘\ ‘*
0 ~,,., 1
‘\. 2
:uts. :llo$
22
24
s 3s
!
22
24
24
Fig. 6. Displacement of [‘251]-E2 from heat-labile and heat-stable receptors at 22°C. For details of this experiment see Text. Addition of radioinert E 2 : open circles and squares; buffer only: closed circles and crosses; (a) and (b) heat-stable, (c) and (d) heat-labile forms; (a) and (c) cytosol, (b) and (d) nuclear homogenate. Crosses and squares indicate a ZOO-foldexcess of DES present during equilibration.
functions in the biological actions of oestrogens. This possibility deserves further investigation.
REFERENCES Hammond K. D., Braunsberg H. and Pflaeger J. E.: Assay and distribution of progesterone receptors in nuclear preparations from human endometrium. ,r. Endocr. 85, (1980) 45~. Hammond K. D. and Braunsberg H.: Assay of human endometrial progesterone, receptors using the natural hormone and a polyethylene glycoi precipitation technique. J. steroid Biochem. 13 (1980) 1147-1156. Clark J. H. and Peck E. J.: Female Sex Sreroids. Receptors and Function. Springer, Berlin (1979). Sica V.. Puca G. A.. Molinari A. M., Buonaguro F. M. and Bresciani F.: Effect of chemical perturbation with
NaSCN. on receptor-estradiol interaction. A new exchange assay at low temperature. Biochemistry 19 (1980) 83-88. Batra S., Grundsell H. and Sjoberg N.-O.: Estradial-i7fi and prog~terone ~~~ntratjons in human endometrium during the menstruai cycle. Conrraception 16 (1977) 217-224. Schmidt-Gollwitzer M., Genz I’., Schmidt-Gollwitzer K., Pollow B. and Pollow K.: Correlation between oestradiol and progesterone receptor levels. 17/I-hydroxysteroid dehydrogenase activity and endometrial tissue levels of oestradiol. oestrone and progesterone in women. In ~~o~errju~ Cancer (Edited by M. G. Brush, R. J. 8. King and R. W. Taylor). Bailiiere Tindall. London (1978). pp. 227-241. Bayard F., Damilano S., Robe1 P. and Baulieu E.-E.: Cytoplasmic and nuclear cstradiol and progesterone receptors in human endometrium. J. c/in. Endocr. Met&. 46 (1978) 635-648.
0022-4731/81/010313-04M2.00m CopyrIght Q 1981 Pergamon Press Ltd
OESTROGEN RECEPTOR COMPONENTS HUMAN ENDOMETRIUM
OF
HANNELORRBRA~JNSBRRGand JULIE E. FTLAEGER Department of Chemical Pathology, St. Mary’s Hospital Medical School, London W2 lPG, England SUMMARY Normal human endometrial tissues contain two distinguishable types of low capacity oestrogen receptor sites. Both types are stable at 4 and 15°C but one is highly labile at 30°C and above. In cytosol preparations both types are stable at 22°C at which temperature oestradiol dissociates from receptor
complexes. In nuclear preparations the more labile form is lost rapidly during exchange at 22°C. The presence of KC1 in nuclear homogenates reduced binding of oestradiol to both forms of receptor.
INTRODUCTION
During an investigation of techniques for receptor analysis it was noted that the low-capacity oestrogen binding sites of normal human endometrial tissue included two distinguishable moieties. The present investigation was undertaken to demonstrate the existence of two species of receptor in cytosol and nuclear preparations, and to examine their relative stability under conditions necessary to effect exchange of endogenous bound steroid. MATERIALS
AND
METHODS
The methods used for tissue fractionation and receptor analysis have been published [l, 23 and are outlined in Figs 1 and 2. Cytosol preparations corresponding to 100 mg tissue per ml buffer were used for experiments with [2,4,6,7-3H}-oestradiol ([‘HI-E,, SA 93 Ci/mmol); further S-fold dilutions were made when 16z-[12’I]-oestradiol ([12sI]-E2, initial SA > lOOOCi/mmol) was used. Washed nuclear pellets [I] were taken up in volumes of buffer corresponding to 400 mg and 100 mg tissue per ml in experiments with C3H]-E2 and [‘251]-E2, respectively. RESULTS
On warming at 40°C. two species of low capacity oestradiol binding sites can be distinguished in preparations from human endometrium (Fig. 3): a labile form which is destroyed within 20 or 30min and a more stable form which is lost more slowly at this temperature. In most samples examined so far the heat-stable form has been present in nuclear homogenates but its proportion has tended to be low in cytosol preparations. Most nuclear preparations contain both forms, but the heat-labile form may be absent in some preparations. Since [3H]-E2 and [1251]-E2 gave similar results, the latter has been used in the present work because of its higher specific activity and the greater sensitivity achievable.
High ionic strength solutions extract nuclear oestradiol receptors incompletely [3], and may preferentially extract one of the two forms of receptor. Figure 4 shows the results of an experiment to examine this possibility. Addition of KCl (final concentration 0.4M) to a nuclear homogenate markedly reduced binding to both receptor moieties. Extraction of intact nuclei with KC1 was incomplete and there appeared to be a loss of both receptors forms. The two types of receptor bound [i2’I]-E2 at simiiarrates (Fig. 5). After 22 h the amounts bound at 4 and at 15°C were similar and there was no evidence of any appreciable loss at these temperatures. At 30°C approximately 5% of the stable form and 16% of the labile form was lost during the first hour of incubation (data not shown). Exchange of bound steroid was therefore studied at lower temperatures. Aliquots (25 ~1) of nuclear homogenate were equilibrated with [ ‘251]-E2 in buffer (25 ~1, final concentration 47 PM) at 4°C for 17 h. A lOO-fold excess of radioinert E2 in 5 ~1 buffer was added to one series of tubes and 5 4 buffer only to another (controls). Dissociation of radiolabelled steroid was followed for a period of 27 h. At 4’C there was no loss of radiolabelled steroid from either type of receptor in the controls and in the presence of radioinert E, (data not shown). Thus both types of binding sites are stable and do not dissociate from receptor at 4°C. Figure 6 shows the results of a similar experiment at 22°C. The cytosol preparation contained only a trace of the more stable receptor species. Both forms appeared to be stable at 22°C and exchange was complete after 22 h. When the nuclear homogenate was used, there was little or no loss of the more stable form of receptor. but binding to the labile form in the controls had decreased appreciably by 22 h. Exchange assays at this temperature would, therefore, lead to losses of the labile form of receptor. The rate of dissociation of Et from calf uterine receptors increases in the presence of sodium thiocyanate [4]. A concentration of 0.5 M sodium thiocyanate caused incomplete precipitation of receptors by 313
HANNELORE BRAUNSBERG and JULIEE. PFLAEGER
314
PREPARATION OF TISSUE FRACTIONS.
RECEPTOR ASSAY
@=-T-ml
cl A
Fig. 1. Preparation of tissue fractions. polyethylene glycol, and there was no evidence of dissociation from human endometrial cytopfasmic or nuclear receptors at 4°C. DISCUSSION
The results presented could perhaps be explained without postulating the existence of two distinct
itif%&?~.
Fig. 2. Assay of heat-labile and heat-stable receptor species. Twenty-five ~1 of cytosol or nuclear preparations were incubated with 25 ~1 buffer containing C3H]-E, or [t2sI]-Et. Non-saturable binding was estimated in the presence of a 200-fold excess of diethylstilboestrol (DES).
receptor types. The decrease in binding of radiolabelled steroid after warming (Fig. 3) might be due to dilution by endogenous steroid released from binding sites. This is unlikely since the magnitude of change
. . CYTOSOL 2n-E2.
\
20 5. cYlosoL
,251~22.
h
Fig. 3. Etfect of warming on receptor binding in cytosol and nuclear preparations. After warming at 40°C for different times, samples were equilibrated at 4°C for 24 h with (a) [‘HI-E,, 1.3 nM. (b) [‘HI-E,, 730pM. (c) [‘2S1]-E,. 93 pM. (d) [*z51]-E2, 45 pM: labelled E, only (0). labelled E2 + DES (0).
Components of endometrial oestrogen receptors
l’r
.
I
.
+DES. 0
4
lo
30
20
40
4. b MJaEM
I,
:
nowGEN4l.E
+ UCI.
0 0
s
-DES.
+oEs. 0
x)
20
1 40
30
4 c. ICI
EXTRACT.
3 2 1 ,b= 01
-0Es. o
___;f
l
+m. 0
20
lo TIME
HEATED.
30 hnhutms)
40
Fig. 4. E&t of KCI (0.4 M) on receptor binding by nuclear homogenate and extraction of whole nuclei with 0.4M KCI. A nuclear suspension was divided into two parts. One part was homogenized and samples were warmed at 40°C for different times after addition of buffer only (a) or of buffer containing KCf (b). Intact nuclei were extracted with KC1 at 4” for 30 min and samples of extract warmed for different times (c). All samples were finally equilibrated with [ rz51]-E1 (1OOpM) at 4°C for 22 h.
would require endogenous levels greater than those reported [S, 63. Another possibility is that the labile and stable forms might represent unbound and bound receptor sites, respectively. The results in Fig. 5 and the fact that no exchange takes place at 4°C make this unlikely. The stable form could result from transformation (activation?) of the labile form. This would only be consistent with results in Figs 3 and 4 if the product contained fewer binding sites than the precursor, a possibility which cannot be excluded. The two forms could represent cytoplasmic (labile and nuclear (stable) receptor species, the presence of both in cytosol or nuclear preparations being due to contamination of one with the other. This possibility is under investigation. but the high proportions of the labile form in some nuclear preparations make it unlikely. That two distinguishable low capacity receptor types may be present in nuclear preparations is further supported by the differences in stability of steroid-bound sites observed at 22°C (Fig. 6) and the difference in loss at 3O’C. The evidence thus supports the existence of two
315
oestrogen receptor forms present in varying proportions in cytosol and nuclear preparations. Methods which only use “saturability” to distinguish receptor sites from “non-specific” binding estimate the sum of the two types. Saturation analysis (data not shown) indicates that both the heat-labile and the heat stable forms have high affinity (Kd of the order of lo-” to lo-i” M) and low capacity. Neither species thus corresponds to Type II receptors [33. The results shown in Fig. 4 demonstrate a loss of nuclear oestrogen receptor binding sites in the presence of KCl. It is possible that similar losses are incurred when KC1 extraction is used to estimate nuclear receptors. Our results differ in several respects from those of Bayard et aI.[7j who also studied human endometrial tissues. In their experiments, cytoplasmic oestrogen receptors appeared to be stable at 30°C whereas we find one form to be highly labile. They also found nuclear receptors to be stable during exchange experiments at 30°C using whole nuclei while our nuclear homogenates contained a form which is highly labile at that temperature. These discrepancies may be due to differences in techniques or in the distribution of receptor species present in the tissues examined. The existence of two distinguishable types of high aflinity binding sites in human endometrial nuclei raises the question as to whether they serve different 15
/I=’
f_________8--9--o
.
lo
:
/
5 /
NDN-3FECIFC
t’
or
*
1
’
2
mNDwG.
*
’
3
4
*’
HEAT-LABILE
I
2
3
4
23
0 .
#I’
t
Y’
22
o/__________”
20
’
21
24
l 0
RECEPTOR.
”
21
22
23
24
TIME. (hours)
Fig. 5. Binding of heat-labile and heat-stable nuclear receptors with [ ‘251]-E2 at 4°C (0) and 15” (0).
316
HANNELORE BRAUNSBERG and JULIE E. PFLAEGER CYTOSOL. ,W” E2 *a-
NUCLEI.
b.
a.
M-
.
-
.
lo
C.
_..__z_________--. .
15
.
D
.t II 0
3
‘. ‘\ ‘*
0 ~,,., 1
‘\. 2
:uts. :llo$
22
24
s 3s
!
22
24
24
Fig. 6. Displacement of [‘251]-E2 from heat-labile and heat-stable receptors at 22°C. For details of this experiment see Text. Addition of radioinert E 2 : open circles and squares; buffer only: closed circles and crosses; (a) and (b) heat-stable, (c) and (d) heat-labile forms; (a) and (c) cytosol, (b) and (d) nuclear homogenate. Crosses and squares indicate a ZOO-foldexcess of DES present during equilibration.
functions in the biological actions of oestrogens. This possibility deserves further investigation.
REFERENCES Hammond K. D., Braunsberg H. and Pflaeger J. E.: Assay and distribution of progesterone receptors in nuclear preparations from human endometrium. ,r. Endocr. 85, (1980) 45~. Hammond K. D. and Braunsberg H.: Assay of human endometrial progesterone, receptors using the natural hormone and a polyethylene glycoi precipitation technique. J. steroid Biochem. 13 (1980) 1147-1156. Clark J. H. and Peck E. J.: Female Sex Sreroids. Receptors and Function. Springer, Berlin (1979). Sica V.. Puca G. A.. Molinari A. M., Buonaguro F. M. and Bresciani F.: Effect of chemical perturbation with
NaSCN. on receptor-estradiol interaction. A new exchange assay at low temperature. Biochemistry 19 (1980) 83-88. Batra S., Grundsell H. and Sjoberg N.-O.: Estradial-i7fi and prog~terone ~~~ntratjons in human endometrium during the menstruai cycle. Conrraception 16 (1977) 217-224. Schmidt-Gollwitzer M., Genz I’., Schmidt-Gollwitzer K., Pollow B. and Pollow K.: Correlation between oestradiol and progesterone receptor levels. 17/I-hydroxysteroid dehydrogenase activity and endometrial tissue levels of oestradiol. oestrone and progesterone in women. In ~~o~errju~ Cancer (Edited by M. G. Brush, R. J. 8. King and R. W. Taylor). Bailiiere Tindall. London (1978). pp. 227-241. Bayard F., Damilano S., Robe1 P. and Baulieu E.-E.: Cytoplasmic and nuclear cstradiol and progesterone receptors in human endometrium. J. c/in. Endocr. Met&. 46 (1978) 635-648.