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Evidence for triiodothyronine receptors in human endometrium and myometrium
Evidence for triiodothyronine receptors in human endometrium and myometrium john L. Kirkland, M.D.,* Venkat Mukku, Ph.D., Marie Hardy, B.S., and Ronald Young, M.D. Houston, Texas Thyroid gland dsyfunction in humans may cause various female reproductive tract disorders. Thyroid hormone action is thought to be mediated by high-affinity low-capacity raceptor proteins located in the nucleus. The sMiles detailed in this report were undertaken to determine if uterine nuclei contain specific high-affinity receptors for thyroid hormone. Nuclei from human endometrium and myometrium were prepared by homogenization and centrifugation following routine SLirgical prOcedures. With the use of isolated nuclei, binding experiments with 1251-triiodothyronine (T3 ) revealed a dissociation constant of approximately 1 x 1o-BM in both endometrium and myometrium with a maximum number of binding sites equivalent to 0.06 and 0.21 pmol/mg of DNA, respectively. The solubilized binding sites were destroyi:KI largely by trypsin treatment. Competition experiments revealed the following relative binding affinities for these nuclear binding sites: L-T3 > D-T3 > L-thyronine >reverse T3 • These results indicate the presence in the human uterus of specific high-affinity binding sites with characteristics expected of a T3 receptor and thus raise the possibility that thyroid hormone may exert effects on the uterus through these raceptors. (AM. J. 0BSTET. GYNECOL. 146:380, 1983.)
The rat uterus has been recognized for many years as an estrogen-sensitive organ. Recently, drug-induced diabetes, 1- 3 hypothyroidism,4 • 5 and hypophysectomyti have been reported to affect selectively specific parameters of estrogen-induced uterine growth and development in the immature rat. Other hormones affecting the uterus include glucocorticoids,1- 9 growth hormone, 10 and androgens.U In humans, deleterious effects of hypothyroidism and hyperthyroidism on the female reproductive tract are well known. In an attempt to understand better the role of thyroid hormone in estrogen-induced uterine growth and development, we have documented the existence of a high-affinity, lowcapacity (dissociation constant [Kd] = 0.6 X w- 9 M) Ta receptor in the rat endometrium arid myometrium. t In view of the clinical pathophysiology of the reproductive tract observed in women with thyroid dysfunction, we decided to determine if T 3 receptors are present in human endometrium and myometrium. This report describes studies which suggest the presence of highaffinity, low-capacity T 3 receptors in the human uterus. From the Departments of Pediatrics and Obstetrics and Gynecology, Baylor College of Medicine, and the Department of Pharmacology, University of Texas Medical School. Supported by National Institutes of'Health Grant HD-08615 and by a grant from the Texas Allergy Research Foundation. Received for publication November 8, 1982. Revised December 13, 1982. Accepted januo.ry 3, 1983. Reprint requests: Dr. john L. Kirkland, Department of Pediatrics, Endocrinology Section, Baylor College of Medicine, Hooston, Texas 77030. *Recipient of a National Institutes of Health Clinical Investigator Award. tMukku, V., Kirkland,]. L., Hardy, M., et al.: Unpublished data.
380
Material and methods
All studies detailed in this report were approved by the Institutional Review Board for Human Research. Fresh endometrium and myometrium from surgical procedures were placed as rapidly as possible in saline at 0° to 4° C and washed. The individual specimens were weighed and frozen until used. All specimens were reported as lacking cancer by histopathologic examination. Unlabeled T 3 and reverse T 3 (r-T 3) were obtained from Calbiochem, thyroxine (T 4 ) was obtained from Sigma Chemical Company, and 125 I-T3 (293 Ci/mmol) was purchased from Abbott Laboratories. Plastic columns (2.1 ml void volume) prepacked with Sephadex G-25 were obtained from Isolabs. All other reagents were the highest grade commercially available. The endometrium was homogenized in an ice-cold Teflon glass homogenizer with five strokes. The myometrium was sliced into small fragments (1 to 2 mm) and homogenized in the same manner. The total amount of tissue homogenized at one time was always less than 200 mg/1.5 mi. The homogenate was filtered through eight layers of nylon mesh. The homogenate was then centrifuged at 800 x g for 10 minutes. The homogenization and assay buffer consisted of 10 mM Tris, 3 mM calcium chloride, 1 mM dithiothreitol, 0.25M sucrose, and 0.1% Nonidet P-40 detergent, with a pH of 7 .6. The T 3 -binding studies were performed by addition of0.2 ml of the nuclear suspension (containing 30 to 60 1-'g of DNA) to 12 by 75 mm tubes containing 125 l-T 3 (at the concentrations indicated in individual experiments) in 0.2 ml of the assay buffer. A 100-fold excess of unla-
Endometrial and myometrial T3 receptors
Volume 146 Number 4
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-Kd= 4.1 xto-9M
n'o,
"'
-
~~0
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4
'
0
2
4
Bmax • 0.21 pmole/mg DNA
\
0
6
BOUND T 3
-
' 'o '
Kd"' 9.7
X
10-10 M
o,
0
-o
o\
0 0
8
o,
'
\
8
/. 10
12
(fmoles)
Fig. 3. T 3 binding as a function of hormone concentration. Aliquots of a suspension of human myometrial nuclei were incubated for 20 hours at 0° C in the presence of the indicated concentrations of 125 I-T 3 (total) or the labeled hormone plus a 100-fold excess cold T 3 (nonspecific). Specific binding was determined by the difference between the two curves.
Table I. Effect of trypsin treatment on . 1'3 binding in extracts of uterine nuclei
'o,
(')
~
~' ""'
nM
Fig. 1. T 3 binding as a function of hormone concentration. Aliquots of a suspension of human endometrial nuclei were incubated for 20 hours at 0° C in the presence of the indicated concentration of 125 1-T3 (total) or the labeled hormone plus a 100-fold excess cold T 3 (nonspecific). Specific binding was determined as the difference between the two curves. 20
~
ID
1.2
0.8
12
.....
• / _...o e_...o
0
(')
[
II.
........... , / 0
:::1
20
TOTAL
381
2
4
BOUND
''
Bmax .. o.o& pmole/mg DNA
'
6
/ 8
10
T 3 (fmoles)
Sample
T 3 bourul (cpm)
Trypsin + trypsin inhibitor Total binding Nonspecific binding Specific binding
5,220 2,490 2,730
Trypsin alone Total binding Nonspecific binding Specific binding
2.285 1,430 8!'15
Specific binding which is trypsin sensitive =
(2,730
855)
~2.~
Fig. 2. Scatchard plot ofT3 binding in the human endometrial nuclei. The line represents the data in Fig. I which was corrected by the Rosenthal method as described in Material and methods.
beled T 3 was used to assess nonspecific binding in parallel samples. Incubations were performed at oo to 4o C for 20 to 24 hours, a length of time shown previously to allow for saturation. Upon completion of the incubation, 0.6 ml of cold assay buffer were added to each tube. Then the tubes were vortexed and centrifuged at 800 x g for l 0 minutes. The supernatant was discarded and the pellets were washed three times with 1 ml of assay buffer and then counted in a Beckman 5000 gamma counter. The DNA content of the final pellet was determined by means of the diphenylamine reaction. 12 The nuclei were extracted with salt as previously described.13 The nuclear pellets prepared as described above were suspended in TEDG buffer (30 mM Tris, I 0 mM ethylenediaminetetraacetic acid [EDTA], 5 mM dithiothreitol, and I 0% glycerol) containing 400 mM
69%.
sodium chloride and 5 mM magnesium chloride, with a pH of 8. The suspension was vortexed every 5 minutes for 45 minutes. This was followed by centrifugation at 20,000 X g for 20 minutes. The resulting supernatant was incubated with an equal volume of TEDG buffer containing 125 1-T3 for 40 hours at 0° to 4° C. Trypsin (l mg/ml) was added to one aliquot of each sample and a combination of trypsin plus soybean trvpsin inhibitor (6 mg/ml) was added to a second aliquot. The incubations were continued overnight at I oo C. Aliquots (0.2 ml) of each sample were placed on prepacked columns of Sephadex G-25 equilibrated with TEDG buffer containing 200 mM sodium chloride and 2.5 mM magnesium chloride. The columns were eluted with the same buffer and the radioactivity was determined in each fraction. Results
Fig. I is an illustration of data from a saturation study with human endometrial nuclei. The total bind-
382
Kirkland et al.
June 15, 1983 Am. J. Obstet. Gynecol.
•
~·
100
CJ
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2i z iii u
~o. ~T4
80
60
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~
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r-T3
A
~~ \
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.
.~.~
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o
11
\
"'
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·~
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'----· •
0--
0.1
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10
100
1000
UNLABELLED COMPETITOR (nM)
Fig. 4. Competition ofT3 binding in the human endometrial nuclei. Samples were incubated at oo C for 20 hours in the presence of 0.5 nM 125 I-T3 plus the indicated concentrations of unlabeled competitors. The maximum level of specific binding (that is, 100% specific binding) was determined by the difference in binding between samples containing 0.5 nM 125 1-T 3 alone or containing a 100-fold excess of unlabeled T 3 •
ing at the indicated concentrations oflabe!ed hormone, the nonspecific binding (in the presence of a 100-fold excess of T 3), and the specific binding are shown. The specific binding appears to be approaching saturation at approximately 1.6 nM. A Scatchard plot with the Rosenthal correction is shown in Fig. 2. 14 • 15 This data revealed a Kd = 9.7 X I0- 10 M and a maximum number of binding sites (Bmaxl = 0.06 pmol of hormone bound per milligram of DNA. Saturation experiments with myometrial nuclei are illustrated in Fig. 3. The Scatchard analysis with the Rosenthal correction yielded a Kd ==' 4.1 x I0- 9 M. 14 • 15 The maximum number of binding sites in the myometrium was equal to 0.21 pmol ofhorm~ne bound/mg of DNA. Next we determined the effect of other thyroid hormones on the binding ofT3 to uterine thyroid hormone receptors. In this experiment (Fig. 4), the abilities of unlabeled L-T 3 , unlabeled D-T3 , unlabeled L-T 4 , and unlabeled r-T 3 to compete with radioactive T 3 are shown. D-T3 had a relative affinity of 40% compared with that ofT 3 , L-T 4 had a relative affinity of2.5%, and r-T3 had a relative affinity of 0.1 %, based on the concentration of competitor required to inhibit the specific binding of labeled T 3 by 50%. We next sought to determine if the binding material present in the endometrium was proteinaceous. This was performed by extracting the nuclear fraction with high salt and then incubating the solubilized extract ·with 1 n~1 125 ! .. T 3 in the presence of trypsin alone or in trypsin plus trypsin inhibitor. Parallel incubations were performed with unlabeled T 3 ( 100-fold excess) to assess nonspecific binding. Macromolecular-bound T 3 was separated from free hormone by Sephadex G-25 col-
umns. The results of these experiments (Table I) illustrate that under the conditions used trypsin treatment destroys approximately 70% of the specific binding of labeled T 3 in the macromolecular fraction.
Comment These studies document the presence of highaffinity T 3 binding sites in human endometrium and myometrium. Their existence suggests that pathophysiology of the human female reproductive tract resulting from abnormalities of thyroid function may be mediated through thyroid hormone binding sites. Our previous ,.vork regarding the effect of hypothy.. roidism on estrogen-induced uterine growth has demonstrated that thyroid hormone may play a permissive role as opposed to a direct role. 4 • 5 Properties of these binding sites suggest that they represent true thyroid hormone receptors. The high affinity of these sites for T 3 (Kd = l X 10-9 M) is comparable to that observed for T 3 receptors in other mammalian cell types. 16- 23 The quantity of high-affinity binding sites in uterine nuclei is considerably less than that observed in most other tissues. The number of binding sites in the uterus (0.06 to 0.21 pmol/mg of DNA) is less than the values of 0.2 to 0.9 pmol for liver nuclei. 23 • 24 The relative binding activity for L-T 3 , L-T4 , D-T 3 , and r-T3 is less than that observed for thyroid hormone receptors in other tissues. 23 Experiments performed with trypsin and trypsin inhibitor suggest that the receptor macromolecule is proteinaceous. In conclusion, human endometrium and human myometrium contain nuclear T 3-binding sites which are comparable to those in other tissues. The sig-
Endometrial and myometrial T 3 receptors
Volume 146 Number 4
nificance of these putative receptors and their possible relation to human reproductive tract pathophysiology in thyroid dysfunction is unknown.
12. 13.
REFERENCES 1. Fowler, D. D., Szego, C. M., and Sloan, S. H.: Curtailment of uterotrophic action of estrogen by impaired histamine liberation in the alloxan diabetic rat: Reversal by insulin and by adrenalectomy, Endocrinology 72:626, 1963. 2. Ekka, E., Dehertogh, R., and Vanderheyden, 1.: Oestradiol 2, 4, 6, 7,-[3 H]17 uptake and subcellular distribution in the uterus of ovariectomized diabetic rat; induction of early protein synthesis,]. Steroid Biochem. 9:833, 1979. 3. Kirkland,]. L., Barrett, G. N., and Stance!, G. M.: Decreased cell division of the uterine luminal of diabetic rats in response of estradiol, Endocrinology 109:316, 1981. 4. Gardner, R. M., Kirkland,]. L., Ireland,]. S., et al.: Regulation of uterine response to estrogen by thyroid hormone, Endocrinology 103:1168, 1978. 5. Kirkland, J. L., Gardner, R. M., Mukku, V. R., et al.: Hormonal control of uterine growth: The effect of hypothyroidism on estrogen-stimulated cell division, Endocrinology 108:2346, 1981. 6. Kirkland, J. L., Gardner, R. M., Ireland, J. S., et al.: The effect of hypophysectomy on the uterine response to estradiol, Endocrinology 101:403, 1977. 7. Campbell, P. S.: The mechanism of inhibition of uterotrophic responses by acute dexamethasone pretreatment, Endocrinology 103:716, 1978. 8. Velando,J. T., Hisaw, F. L., and Bever, A. T.: Inhibitory action of deoxycortitosterone acetate, cortisone acetate, and testosterone on uterine growth induced by estradiol-! 7, Endocrinology 59:165, 1956. 9. Spaziani, E., and Szego, C. M.: The influence of estradiol and cortisol on uterine histamine of the ovariectomized rat, Endocrinology 63:669, 1958. 10. Grattarola, R., and Li, C. H.: Effect of growth hormone and its combination with estradiol-17 on the uterus of hypophysectomized and hypophysectomized-ovariectomized rats, Endocrinology 65:802, 1959. 11. Schmidt, W. N., and Katzenellenbogen, B. S.: Androgen-uterine interactions: An assessment of androgen interaction with the testosterone-and estrogen-receptor sys-
14. 15. 16. 17.
18.
19.
20.
21. 22. 23. 24.
383
terns and stimulation of uterine growth and progesterone receptor synthesis, Mol. Cell. Endocrinol. 15:91, 1979. Burton, K.: Study of conditions and mechanism of diphenylamine reaction for colorimetric estimation of deoxyribonucleic acid, Biochem. J. 62:315, 1956. Seelig, S., Schwartz, H. L., and Oppenheimer,]. H.: Limitations on the conventional analysis of the interaction of triiodothyronine with solubilized nuclear receptor sites, J. Bioi. Chern. 256:2154, 1981. Scatchard, G.: The attractions of proteins for small molecules and ions, Ann. N.Y. Acad. Sci. 51:660, 1949. Rosenthal, H. E.: A graphic method for the determination and presentation of binding parameters in a complex system, Anal. Biochem. 20:525, 1967. Oppenheimer, J. H., Schwartz, H. L., Surks, M. L, et al.: Nuclear receptors and initiation of thyroid hormone action, Recent Prog. Horm. Res. 32:529, 1976. Samuels, H. H.: In vitro studies on thyroid hormone re· ceptors, in Birnbaumer, L., and O'Malley, B. W.: Receptors and Hormone Action, vol. 3, New York, 1978, Academic Press, Inc., pp. 35-74. Eberhardt, N. L., Apriletti, J. W., and Baxter, J. D.: The molecular biology of thyroid hormone action, in Litwack, G., editor: Biochemical Actions of Hormones, New York, 1980, vol. 3, Academic Press, Inc., pp. 333-344. Latham, K. R., Ring,]. C., and Baxter,]. D.: Solubilized nuclear receptors for thyroid hormones. Physical characteristics and binding properties, evidence for multiple forms, J. Bioi. Chern. 251:7388, 1976. Eberhardt, N. L., Valcana, T., and Timiras, P. S.: Triiodothyronine nuclear receptors: An in vitro comparison of the binding of triiodothyronine to nuclei of adult rat liver, cerebral hemisphere and anterior pituitary, Endocrinology 102:556, 1978. Lindenberg, J. A., Brehier, A., and Ballard, D. L.: Triiodothyronine nuclear binding in fetal and adult lung and cultured lung cells, Endocrinology 103:1725, 1978. Koerner. D., Schwatz, H. L., Surks, M. I.. et al.: Binding of selected iodothyronine analogs to receptor sites of isolated rat hepatic nuclei,J. Bioi. Chern. 250:6417, 1975. DeGroot, L. J., and Torresani,J.: Triiodothyronine binding to isolated liver cell nuclei. Endocrinology 96:357, 1975. Oppenheimer, J. H., Schwatz, H. L.., and Surks, M. 1.: Tissue differences in the concentration of triiodothyronine nuclear binding sites in the rat: Liver, kidney, pituitary, heart, brain, spleen and testis, Endocrinology 95: 897, 1974.
The rat uterus has been recognized for many years as an estrogen-sensitive organ. Recently, drug-induced diabetes, 1- 3 hypothyroidism,4 • 5 and hypophysectomyti have been reported to affect selectively specific parameters of estrogen-induced uterine growth and development in the immature rat. Other hormones affecting the uterus include glucocorticoids,1- 9 growth hormone, 10 and androgens.U In humans, deleterious effects of hypothyroidism and hyperthyroidism on the female reproductive tract are well known. In an attempt to understand better the role of thyroid hormone in estrogen-induced uterine growth and development, we have documented the existence of a high-affinity, lowcapacity (dissociation constant [Kd] = 0.6 X w- 9 M) Ta receptor in the rat endometrium arid myometrium. t In view of the clinical pathophysiology of the reproductive tract observed in women with thyroid dysfunction, we decided to determine if T 3 receptors are present in human endometrium and myometrium. This report describes studies which suggest the presence of highaffinity, low-capacity T 3 receptors in the human uterus. From the Departments of Pediatrics and Obstetrics and Gynecology, Baylor College of Medicine, and the Department of Pharmacology, University of Texas Medical School. Supported by National Institutes of'Health Grant HD-08615 and by a grant from the Texas Allergy Research Foundation. Received for publication November 8, 1982. Revised December 13, 1982. Accepted januo.ry 3, 1983. Reprint requests: Dr. john L. Kirkland, Department of Pediatrics, Endocrinology Section, Baylor College of Medicine, Hooston, Texas 77030. *Recipient of a National Institutes of Health Clinical Investigator Award. tMukku, V., Kirkland,]. L., Hardy, M., et al.: Unpublished data.
380
Material and methods
All studies detailed in this report were approved by the Institutional Review Board for Human Research. Fresh endometrium and myometrium from surgical procedures were placed as rapidly as possible in saline at 0° to 4° C and washed. The individual specimens were weighed and frozen until used. All specimens were reported as lacking cancer by histopathologic examination. Unlabeled T 3 and reverse T 3 (r-T 3) were obtained from Calbiochem, thyroxine (T 4 ) was obtained from Sigma Chemical Company, and 125 I-T3 (293 Ci/mmol) was purchased from Abbott Laboratories. Plastic columns (2.1 ml void volume) prepacked with Sephadex G-25 were obtained from Isolabs. All other reagents were the highest grade commercially available. The endometrium was homogenized in an ice-cold Teflon glass homogenizer with five strokes. The myometrium was sliced into small fragments (1 to 2 mm) and homogenized in the same manner. The total amount of tissue homogenized at one time was always less than 200 mg/1.5 mi. The homogenate was filtered through eight layers of nylon mesh. The homogenate was then centrifuged at 800 x g for 10 minutes. The homogenization and assay buffer consisted of 10 mM Tris, 3 mM calcium chloride, 1 mM dithiothreitol, 0.25M sucrose, and 0.1% Nonidet P-40 detergent, with a pH of 7 .6. The T 3 -binding studies were performed by addition of0.2 ml of the nuclear suspension (containing 30 to 60 1-'g of DNA) to 12 by 75 mm tubes containing 125 l-T 3 (at the concentrations indicated in individual experiments) in 0.2 ml of the assay buffer. A 100-fold excess of unla-
Endometrial and myometrial T3 receptors
Volume 146 Number 4
6
5
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~
24
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4
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0
ID
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OA
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2A
16
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8
.....
4
..
Ill
-Kd= 4.1 xto-9M
n'o,
"'
-
~~0
'
4
'
0
2
4
Bmax • 0.21 pmole/mg DNA
\
0
6
BOUND T 3
-
' 'o '
Kd"' 9.7
X
10-10 M
o,
0
-o
o\
0 0
8
o,
'
\
8
/. 10
12
(fmoles)
Fig. 3. T 3 binding as a function of hormone concentration. Aliquots of a suspension of human myometrial nuclei were incubated for 20 hours at 0° C in the presence of the indicated concentrations of 125 I-T 3 (total) or the labeled hormone plus a 100-fold excess cold T 3 (nonspecific). Specific binding was determined by the difference between the two curves.
Table I. Effect of trypsin treatment on . 1'3 binding in extracts of uterine nuclei
'o,
(')
~
~' ""'
nM
Fig. 1. T 3 binding as a function of hormone concentration. Aliquots of a suspension of human endometrial nuclei were incubated for 20 hours at 0° C in the presence of the indicated concentration of 125 1-T3 (total) or the labeled hormone plus a 100-fold excess cold T 3 (nonspecific). Specific binding was determined as the difference between the two curves. 20
~
ID
1.2
0.8
12
.....
• / _...o e_...o
0
(')
[
II.
........... , / 0
:::1
20
TOTAL
381
2
4
BOUND
''
Bmax .. o.o& pmole/mg DNA
'
6
/ 8
10
T 3 (fmoles)
Sample
T 3 bourul (cpm)
Trypsin + trypsin inhibitor Total binding Nonspecific binding Specific binding
5,220 2,490 2,730
Trypsin alone Total binding Nonspecific binding Specific binding
2.285 1,430 8!'15
Specific binding which is trypsin sensitive =
(2,730
855)
~2.~
Fig. 2. Scatchard plot ofT3 binding in the human endometrial nuclei. The line represents the data in Fig. I which was corrected by the Rosenthal method as described in Material and methods.
beled T 3 was used to assess nonspecific binding in parallel samples. Incubations were performed at oo to 4o C for 20 to 24 hours, a length of time shown previously to allow for saturation. Upon completion of the incubation, 0.6 ml of cold assay buffer were added to each tube. Then the tubes were vortexed and centrifuged at 800 x g for l 0 minutes. The supernatant was discarded and the pellets were washed three times with 1 ml of assay buffer and then counted in a Beckman 5000 gamma counter. The DNA content of the final pellet was determined by means of the diphenylamine reaction. 12 The nuclei were extracted with salt as previously described.13 The nuclear pellets prepared as described above were suspended in TEDG buffer (30 mM Tris, I 0 mM ethylenediaminetetraacetic acid [EDTA], 5 mM dithiothreitol, and I 0% glycerol) containing 400 mM
69%.
sodium chloride and 5 mM magnesium chloride, with a pH of 8. The suspension was vortexed every 5 minutes for 45 minutes. This was followed by centrifugation at 20,000 X g for 20 minutes. The resulting supernatant was incubated with an equal volume of TEDG buffer containing 125 1-T3 for 40 hours at 0° to 4° C. Trypsin (l mg/ml) was added to one aliquot of each sample and a combination of trypsin plus soybean trvpsin inhibitor (6 mg/ml) was added to a second aliquot. The incubations were continued overnight at I oo C. Aliquots (0.2 ml) of each sample were placed on prepacked columns of Sephadex G-25 equilibrated with TEDG buffer containing 200 mM sodium chloride and 2.5 mM magnesium chloride. The columns were eluted with the same buffer and the radioactivity was determined in each fraction. Results
Fig. I is an illustration of data from a saturation study with human endometrial nuclei. The total bind-
382
Kirkland et al.
June 15, 1983 Am. J. Obstet. Gynecol.
•
~·
100
CJ
z
2i z iii u
~o. ~T4
80
60
(J
40
a.
L-T3
0
~
~
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A
~~ \
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.
.~.~
0
o
11
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·~
0~
'----· •
0--
0.1
1.0
10
100
1000
UNLABELLED COMPETITOR (nM)
Fig. 4. Competition ofT3 binding in the human endometrial nuclei. Samples were incubated at oo C for 20 hours in the presence of 0.5 nM 125 I-T3 plus the indicated concentrations of unlabeled competitors. The maximum level of specific binding (that is, 100% specific binding) was determined by the difference in binding between samples containing 0.5 nM 125 1-T 3 alone or containing a 100-fold excess of unlabeled T 3 •
ing at the indicated concentrations oflabe!ed hormone, the nonspecific binding (in the presence of a 100-fold excess of T 3), and the specific binding are shown. The specific binding appears to be approaching saturation at approximately 1.6 nM. A Scatchard plot with the Rosenthal correction is shown in Fig. 2. 14 • 15 This data revealed a Kd = 9.7 X I0- 10 M and a maximum number of binding sites (Bmaxl = 0.06 pmol of hormone bound per milligram of DNA. Saturation experiments with myometrial nuclei are illustrated in Fig. 3. The Scatchard analysis with the Rosenthal correction yielded a Kd ==' 4.1 x I0- 9 M. 14 • 15 The maximum number of binding sites in the myometrium was equal to 0.21 pmol ofhorm~ne bound/mg of DNA. Next we determined the effect of other thyroid hormones on the binding ofT3 to uterine thyroid hormone receptors. In this experiment (Fig. 4), the abilities of unlabeled L-T 3 , unlabeled D-T3 , unlabeled L-T 4 , and unlabeled r-T 3 to compete with radioactive T 3 are shown. D-T3 had a relative affinity of 40% compared with that ofT 3 , L-T 4 had a relative affinity of2.5%, and r-T3 had a relative affinity of 0.1 %, based on the concentration of competitor required to inhibit the specific binding of labeled T 3 by 50%. We next sought to determine if the binding material present in the endometrium was proteinaceous. This was performed by extracting the nuclear fraction with high salt and then incubating the solubilized extract ·with 1 n~1 125 ! .. T 3 in the presence of trypsin alone or in trypsin plus trypsin inhibitor. Parallel incubations were performed with unlabeled T 3 ( 100-fold excess) to assess nonspecific binding. Macromolecular-bound T 3 was separated from free hormone by Sephadex G-25 col-
umns. The results of these experiments (Table I) illustrate that under the conditions used trypsin treatment destroys approximately 70% of the specific binding of labeled T 3 in the macromolecular fraction.
Comment These studies document the presence of highaffinity T 3 binding sites in human endometrium and myometrium. Their existence suggests that pathophysiology of the human female reproductive tract resulting from abnormalities of thyroid function may be mediated through thyroid hormone binding sites. Our previous ,.vork regarding the effect of hypothy.. roidism on estrogen-induced uterine growth has demonstrated that thyroid hormone may play a permissive role as opposed to a direct role. 4 • 5 Properties of these binding sites suggest that they represent true thyroid hormone receptors. The high affinity of these sites for T 3 (Kd = l X 10-9 M) is comparable to that observed for T 3 receptors in other mammalian cell types. 16- 23 The quantity of high-affinity binding sites in uterine nuclei is considerably less than that observed in most other tissues. The number of binding sites in the uterus (0.06 to 0.21 pmol/mg of DNA) is less than the values of 0.2 to 0.9 pmol for liver nuclei. 23 • 24 The relative binding activity for L-T 3 , L-T4 , D-T 3 , and r-T3 is less than that observed for thyroid hormone receptors in other tissues. 23 Experiments performed with trypsin and trypsin inhibitor suggest that the receptor macromolecule is proteinaceous. In conclusion, human endometrium and human myometrium contain nuclear T 3-binding sites which are comparable to those in other tissues. The sig-
Endometrial and myometrial T 3 receptors
Volume 146 Number 4
nificance of these putative receptors and their possible relation to human reproductive tract pathophysiology in thyroid dysfunction is unknown.
12. 13.
REFERENCES 1. Fowler, D. D., Szego, C. M., and Sloan, S. H.: Curtailment of uterotrophic action of estrogen by impaired histamine liberation in the alloxan diabetic rat: Reversal by insulin and by adrenalectomy, Endocrinology 72:626, 1963. 2. Ekka, E., Dehertogh, R., and Vanderheyden, 1.: Oestradiol 2, 4, 6, 7,-[3 H]17 uptake and subcellular distribution in the uterus of ovariectomized diabetic rat; induction of early protein synthesis,]. Steroid Biochem. 9:833, 1979. 3. Kirkland,]. L., Barrett, G. N., and Stance!, G. M.: Decreased cell division of the uterine luminal of diabetic rats in response of estradiol, Endocrinology 109:316, 1981. 4. Gardner, R. M., Kirkland,]. L., Ireland,]. S., et al.: Regulation of uterine response to estrogen by thyroid hormone, Endocrinology 103:1168, 1978. 5. Kirkland, J. L., Gardner, R. M., Mukku, V. R., et al.: Hormonal control of uterine growth: The effect of hypothyroidism on estrogen-stimulated cell division, Endocrinology 108:2346, 1981. 6. Kirkland, J. L., Gardner, R. M., Ireland, J. S., et al.: The effect of hypophysectomy on the uterine response to estradiol, Endocrinology 101:403, 1977. 7. Campbell, P. S.: The mechanism of inhibition of uterotrophic responses by acute dexamethasone pretreatment, Endocrinology 103:716, 1978. 8. Velando,J. T., Hisaw, F. L., and Bever, A. T.: Inhibitory action of deoxycortitosterone acetate, cortisone acetate, and testosterone on uterine growth induced by estradiol-! 7, Endocrinology 59:165, 1956. 9. Spaziani, E., and Szego, C. M.: The influence of estradiol and cortisol on uterine histamine of the ovariectomized rat, Endocrinology 63:669, 1958. 10. Grattarola, R., and Li, C. H.: Effect of growth hormone and its combination with estradiol-17 on the uterus of hypophysectomized and hypophysectomized-ovariectomized rats, Endocrinology 65:802, 1959. 11. Schmidt, W. N., and Katzenellenbogen, B. S.: Androgen-uterine interactions: An assessment of androgen interaction with the testosterone-and estrogen-receptor sys-
14. 15. 16. 17.
18.
19.
20.
21. 22. 23. 24.
383
terns and stimulation of uterine growth and progesterone receptor synthesis, Mol. Cell. Endocrinol. 15:91, 1979. Burton, K.: Study of conditions and mechanism of diphenylamine reaction for colorimetric estimation of deoxyribonucleic acid, Biochem. J. 62:315, 1956. Seelig, S., Schwartz, H. L., and Oppenheimer,]. H.: Limitations on the conventional analysis of the interaction of triiodothyronine with solubilized nuclear receptor sites, J. Bioi. Chern. 256:2154, 1981. Scatchard, G.: The attractions of proteins for small molecules and ions, Ann. N.Y. Acad. Sci. 51:660, 1949. Rosenthal, H. E.: A graphic method for the determination and presentation of binding parameters in a complex system, Anal. Biochem. 20:525, 1967. Oppenheimer, J. H., Schwartz, H. L., Surks, M. L, et al.: Nuclear receptors and initiation of thyroid hormone action, Recent Prog. Horm. Res. 32:529, 1976. Samuels, H. H.: In vitro studies on thyroid hormone re· ceptors, in Birnbaumer, L., and O'Malley, B. W.: Receptors and Hormone Action, vol. 3, New York, 1978, Academic Press, Inc., pp. 35-74. Eberhardt, N. L., Apriletti, J. W., and Baxter, J. D.: The molecular biology of thyroid hormone action, in Litwack, G., editor: Biochemical Actions of Hormones, New York, 1980, vol. 3, Academic Press, Inc., pp. 333-344. Latham, K. R., Ring,]. C., and Baxter,]. D.: Solubilized nuclear receptors for thyroid hormones. Physical characteristics and binding properties, evidence for multiple forms, J. Bioi. Chern. 251:7388, 1976. Eberhardt, N. L., Valcana, T., and Timiras, P. S.: Triiodothyronine nuclear receptors: An in vitro comparison of the binding of triiodothyronine to nuclei of adult rat liver, cerebral hemisphere and anterior pituitary, Endocrinology 102:556, 1978. Lindenberg, J. A., Brehier, A., and Ballard, D. L.: Triiodothyronine nuclear binding in fetal and adult lung and cultured lung cells, Endocrinology 103:1725, 1978. Koerner. D., Schwatz, H. L., Surks, M. I.. et al.: Binding of selected iodothyronine analogs to receptor sites of isolated rat hepatic nuclei,J. Bioi. Chern. 250:6417, 1975. DeGroot, L. J., and Torresani,J.: Triiodothyronine binding to isolated liver cell nuclei. Endocrinology 96:357, 1975. Oppenheimer, J. H., Schwatz, H. L.., and Surks, M. 1.: Tissue differences in the concentration of triiodothyronine nuclear binding sites in the rat: Liver, kidney, pituitary, heart, brain, spleen and testis, Endocrinology 95: 897, 1974.