0013-7227/78/1034-1164$02.00/0 Endocrinology Copyright © 1978 by The Endocrine Society
Vol. 103, No. 4 Printed in U.S.A.
Regulation of the Uterine Response to Estrogen by Thyroid Hormone* RUSSELL M. GARDNERS JOHN L. KIRKLAND, JUDY S. IRELAND, AND GEORGE M. STANCEL^ Departments of Pharmacology and Pediatrics, The University of Texas Medical School at Houston, Houston, Texas 77025 ABSTRACT. After estradiol (E2) administration, early increases (within 4 h) in uterine wet weight and the synthesis of 2-deoxyglucose-6-phosphate from 2-deoxyglucose are similar in ovariectomized rats and in ovariectomized rats made hypothyroid by feeding a low iodine diet containing propylthiouracil. Most late uterine responses occurring 24 h after E2 treatment, however, are greatly diminished in the hypothyroid animals. The diminished responses include increases in uterine wet weight, dry weight, protein content, RNA content, and the incorporation of thymidine into uterine DNA. One response, the synthesis of 2-deoxyglucose-6-phosphate from 2-deoxyglucose is not diminished in hypothyroid rats 24 h after E2 treatment. The diminished uterine response is not due to a shift in the dose-response curve
for E2, but results from a decrease in the magnitude of the maximum uterine response. Treatment of hypothyroid rats with exogenous T,-i restores the diminished uterine response in a dose-dependent and time-dependent manner. A dose of 0.5 /xg T:1/100 g BW for 5 days restores the response completely, while 48-72 h of treatment with higher doses of T.i are required to restore the response. The effect of T.) is not mediated by the pituitary, since exogenous T.i restores diminished uterine responses in ovariectomized, hypophysectomized animals. These results suggest that thyroid hormones may have a direct effect on the uterus which regulates the responsiveness of the organ to E2. (Endocrinology 103: 1164, 1978)
T
HE OVERALL response of the rat uterus to estrogens is a complex process which seems to involve at least two major temporal phases, an early phase and a late phase (1-5). Specific responses occurring in the early phase are increases in uterine wet weight (3, 6, 7), glucose metabolism (7-9), and RNA polymerase activities (10-13), while late responses include increases in uterine dry weight (4-6), DNA synthesis (9, 14), protein (10, 15), and RNA content (10, 15). It has been suggested that these late uterine responses represent true growth of the organ, while the early responses represent a preparation for this
growth. The complete uterine response seems to be mediated by a series of interactions in which hormonal regulation may exist at multiple sites within uterine nuclei and at various times after hormone treatment (see References 5 and 16 for recent reviews). In previous work we observed that hypophysectomy could selectively diminish most late uterine responses to estrogen occurring 24 h after hormone treatment, but had little effect on uterine responses occurring within 4 h after estrogen administration (17). Therefore, we initiated a series of studies to determine which specific pituitary factor(s) was responsible for the decreased uterine response observed in hypophysectomized animals. Received October 21, 1977. Address requests for reprints to: Dr. George M. StanIn this study we have observed that thyroid eel, University of Texas, Department of Pharmacology, hormones regulate the uterine response to Health Sciences Center, P.O. Box 20708,6400 West Cullen estrogens. Most late uterine responses to esStreet, Houston, Texas 77025. * This work was supported by NIH Grant HD-08615. trogens are markedly decreased in hypothyA preliminary report of this investigation was presented roid animals, and this effect is reversed by the at the Federation of American Societies for Experimental Biology, Chicago, IL, April 1977 (FedProc 36: 368,1977). administration of exogenous T3 in a dose-def Recipient of NIH Research Fellowship Award AM- pendent and time-dependent manner. Exoge05246. Present address: Department of Biology, Rochester nous T also restores the diminished uterine 3 Institute of Technology, Rochester, New York. response to estrogen previously observed in $ Recipient of NIH Research Career Development hypophysectomized animals (17). These reAward 1 K04 HD-00099. 1164 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 28 November 2015. at 00:23 For personal use only. No other uses without permission. . All rights reserved.
THYROID REGULATION OF ESTROGEN ACTION
suits indicate that the decreased uterine response observed in hypophysectomized animals results from hypothyroidism secondary to decreased levels of TSH, and further suggest that thyroid hormones may have a direct effect on the uterus which regulates the responsiveness of this organ to estrogens. Materials and Methods Rats used for the majority of studies in this work were obtained from Texas Inbred Mouse Co., Houston, TX. All animals used in this work were ovariectomized at 22-24 days of age (approximately 50 g BW) and were allowed to recover for 1-2 days. Animals were then fed ad libitum either a diet of normal rat chow or a low iodine diet containing 0.15% propylthiouracil (PTU) obtained from Teklad, Madison, WI, and given drinking water for 30 days before use. At this time, animal weights ranged from 156-225 g in the group receiving the normal diet, and from 64-90 g in those receiving the PTU diet. Serum T 4 levels were also measured at this time by RIA of serum from randomly selected rats in both groups. These measurements were performed by Veterinary Laboratory Associates, Houston, TX. Mean serum T4 levels for the normal group were 3.46 /xg/100 ml ± 0.19 (n = 11) and for the PTU groups were 0.58 jug/100 ml ± 0.08 (n = 9). For studies with hypophysectomized animals, either ovariectomized (OVX) or ovariectomized, hypophysectomized (OVX-HX) rats were purchased from Zivic-Miller Laboratories, Allison Park, PA, and maintained as previously described (17). Before sacrifice, animals were injected ip with the doses of 17/S-estradiol (Schwarz/Mann) indicated in the text for individual experiments or with the vehicle alone (control groups). Estradiol was routinely administered in 0.5 ml 95% saline-5% EtOH, except in the dose-response studies where 0.5 ml 50% saline-50% propylene glycol was used. Animals were sacrificed by decapitation and the uteri were removed, stripped of adhering fat and mesentery, and weighed on a Cahn Electrobalance. Uterine dry weights were measured with a Mettler Analytical Balance after drying (90 C) to constant weight. Uterine DNA synthesis was measured as described by Kaye et al. (14). Uteri were incubated in Eagle's HeLa medium (Difco) for 1 h at 37 C with 0.25 juCi [3H]thymidine (Schwarz/Mann)/ml and a total thymidine concentration of 4 X 10"b M. Incubations were performed under 95% O2-5% CO2 with
1165
gentle shaking. After the incubation the uteri were homogenized, and nucleic acids and proteins were precipitated by perchloric acid (PCA; 0.25 N) at 0 C. After washing, the pellets were hydrolyzed at 90 C for 30 min in the presence of 0.4 N PCA. Aliquots of the final supernatants were then counted to dtermine [3H]thymidine incorporation. The conversion of 2-deoxyglucose to 2-deoxyglucose-6-phosphate was measured as described by Gorski and Raker (18). Uteri were incubated in Eagle's HeLa medium containing 0.5 juCi/ml 2-[l14 C]deoxyglucose (New England Nuclear) for 30 min at 37 C under an atmosphere of 95% O2-5% CO2. After homogenization and removal of PCAinsoluble material, the samples were adjusted to alkaline pH (approximately 8.0) and insoluble potassium perchlorate was removed by centrifugation. The I4C-labeled 2-deoxyglucose-6-phosphate formed was then absorbed with Bio-Rad Ag2-X8 resin in a batch procedure, the resin was washed three times with 4.0 ml distilled water, and then extraction was performed with HC1 (0.2 N) to remove the 2-deoxyglucose-6-phosphate for radioactive counting. Protein content was measured by a modification (19) of the method of Lowry et al, and RNA content was measured by the orcinol reaction (20). Where indicated in the text, L-T 3 (Sigma) was administered sc in dilute base at the dose levels and times described for individual experiments. For studies of the retention patterns of nuclear estrogen receptor complexes, animals were injected ip with 0.2 jug [3H]estradiol (New England Nuclear)/100 g BW (SA, 10-15 Ci/mmol). In parallel experiments, animals received the same dose of [3H]estradiol plus a 100-fold excess of unlabeled hormone to correct for nonspecific binding. At the times indicated in the text, animals were sacrificed, uterine nuclei were prepared and washed, and [3H]estradiol was extracted from the nuclei with 95% EtOH, as previously described (21). All values indicated in the text and figures represent specific binding. Serum GH levels were kindly measured by Dr. Max Hutchins, Department of Physiology, The University of Texas Dental School, Houston, TX, using RIA, as described previously (22).
Results The data in Fig. 1 illustrate the uterine response at various times after estrogen administration to normal and hypothyroid rats. Both groups of animals show an initial increase in wet weight at 4 h, which is compa-
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GARDNER ET
1166
Endo , 1978 Vol 103 , No 4
AL.
In addition to the data in Fig. 1, other uterine responses occurring 24 h after estrogen administration are also greatly diminished in hypothyroid animals. These results are illustrated in Table 1. Since we had previously observed that hyT —T pophysectomy did not appreciably diminish ^ T — 60 NORMAL estrogen-stimulated uterine glucose metabo/I 40 lism (17), it was next of interest to study this 2 I -o parameter in hypothyroid animals. The reE o 20 ' PTU sults of these studies are depicted in Table 2, si 0 which illustrates the effect of estrogen treatment on the synthesis of 2-deoxyglucose-6ZbU phosphate from 2-deoxyglucose 4 and 24 h 220 after treatment in normal and hypothyroid animals. Unlike the other parameters studied, i 180 2-deoxyglucose-6-phosphate synthesis is not NORMAL 140 decreased in hypothyroid animals. We next determined whether the decreased PTU 100 response observed in hypothyroid animals 24 5 h after estrogen treatment (Fig. 1) was due to a decrease in the maximum uterine response 12 24 36 0 4 TIME (Hours) or a shift in the dose-response curve for estraFIG. 1. Time course of estradiol (E2) action in normal diol. The results of studies designed to differand hypothyroid (PTU) animals. Animals were treated entiate these two possibilities are illustrated with E2 (4 jug/100 g BW) at the indicated times before in Fig. 2. This figure reveals that the uterine sacrifice. Saline-treated control values did not change response to estrogen is maximum at 0.4 jug over the time course shown (data not shown). Upper panel, Uterine wet weight values; lower panel, responses estradiol/100 g BW in both groups, and that as percentages of zero time controls. Values represent larger doses of the hormone fail to restore the uterine response in hypothyroid animals. means with the indicated SEM (n = 6-7/point). rable relative to saline-treated controls in both groups. However, at longer times, e.g. 24 h, the response is clearly diminished in the hypothyroid animals.
h ,
f'
•'•---6
^
TABLE 1. Effect of hypothyroidism on the uterine response to estradiol (E2) 24 h after treatment Treatment Parameter Uterine dry weight (mg/uterus) Normal PTU Uterine protein content (mg/uterus) Normal PTU Uterine RNA content (/xg/uterus) Normal PTU Uterine DNA synthesis (cpm [:)H]thymidine incorporated//^ DNA) Normal PTU
Response as % of saline control
Saline
E2
4.25 ± 0.27 (6) 2.74 ± 0.14 (7)
6.42 ± 0.27 (6) 2.96 ± 0.27 (7)
151 ± 6 108 ± 10
2.87 ± 0.21 (7) 2.15 ± 0.09 (7)
5.18 ± 0.37 (7) 2.82 ± 0.13 (7)
180 ± 13 131 ± 6
91.6 ± 10 (7) 92.0 ± 9.4 (7)
240 ± 27 (7) 128 ± 18 (7)
262 ± 29 139 ± 19
30.5 ± 1.9 (7) 22.0 ± 0.7 (7)
208 ± 51 (7) 39.5 ± 2.3 (7)
682 ± 168 180 ± 11
Animals were mantained on a normal diet (Normal) or a PTU-containing diet, as described in Materials and Methods. Animals were then treated with E2 (4 jug/100 g BW) or the vehicle alone (saline) 24 h before sacrifice. All values represent means ± SEM; the number of animals used for each determination is in parentheses. E2 treatment produced significant increases (P < 0.05) in all parameters for both groups of animals, except for dry weight and RNA content in the hypothyroid (PTU) animals; but increases produced by E2 treatment (response as a percentage of saline control) in the normal animals are significantly different {P < 0.005) from those produced in hypothyroid animals for all parameters.
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THYROID REGULATION OF ESTROGEN ACTION TABLE 2. Effect of estradiol (E2) treatment on 2-deoxyglucose-6-phosphate synthesis in normal and hypothyroid rats Treatment
Re-
Saline
E,
sponse as % of saline control
PTU
1132 ± 42 (4) 1074 ± 48 (5)
2028 ±246 (6) 1940 ± 9 1 (6)
179 ± 22 181 ± 9
24 H Normal PTU
714 ± 103 (5) 547 ± 36 (6)
1238 ± 147 (7) 966 ± 7 0 (7)
173 ± 21 177 ± 13
Time of sacrifice 4H Normal
Animals were fed a normal diet (Normal) or a PTUcontaining diet, as described in Materials and Methods. Either 4 or 24 h before sacrifice, animals were treated with 4 /xg/100 g BW E2 or the vehicle alone (saline). The synthesis of 2-deoxyglucose-6-phosphate from 2-deoxyglucose was then measured, as described in Materials and Methods. Values (counts per min per uterus) represent means ± SEM; the number of animals in each group is given in parentheses.
1167
In order to insure that the decreased long term uterine responses observed in the preceding studies were due to decreased levels of thyroid hormones, it was important to demonstrate that the observed effects could be reversed by treatment with exogenous thyroid hormone. The data in Fig. 3 thus illustrate the effect of T3 administration on the uterine response to estradiol in hypothyroid rats. These results illustrate several salient features. First, T 3 alone does not elicit any uterine response (Fig. 3, upper panel: saline controls). Second, T 3 administration to hypothyroid animals restores the ability of the uterus to respond to estradiol 24 h after treatment. A dose of 0.5 jug T3/IOO g BW restores the response completely, and a dose of 0.05 jug T 3 is approximately half-maximal. The dose-response curves for T 3 restoration of estrogen-induced increases in uterine protein and RNA content
60 50 40
a. O
si
•»—T"* 20 25 0 SALINE 200 180
/I
160
NORMAL
1
140
/I
180 I
.—•I
120
140
PTU 0 .004
220
.04
DOSE E 2
.4
4
40
Uig/100 g B.W.)
FIG. 2. Estradiol (E2) dose-response curves for normal and hypothyroid (PTU) animals. Animals were treated with the indicated doses of E2 24 h before sacrifice. Upper panel, Uterine wet weight values; lower panel, responses plotted as percentages of control (animals not receiving E2). Values represent means with the indicated SEM (n = 5-8/point).
Dose-response studies for increases in other uterine parameters, e.g. uterine protein and RNA content, 24 h after hormone treatment were similar to those observed in Fig. 2.
100 0
0.05 DOSE T 3
0.5
5.0
50.0
(iig/100 g B.W.)
FIG. 3. Effect of T:J on the uterine response to estradiol (E2) in hypothyroid animals. Hypothyroid animals were treated daily with the indicated total dose of T3 for 5 days before sacrifice. Twenty-four hours before sacrifice animals at each dose level of T3 were treated with either saline {upper panel, ) or 4 jug/100 g BW E 2 (upper panel, ). Lower panel, Responses as percentages of control animals not receiving T3. The open square near the ordinate (lower panel) represents the response of normal animals to E2 relative to appropriate salinetreated controls. Values represent means with the indicated SEM (n = 6-7/point).
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1168
GARDNER ET AL.
24 h after treatment are also similar. The full restoration of the uterine response to estrogen requires 2-3 days, as illustrated in Fig. 4. For these studies a large dose of T3 (50 jug/100 g BW) was used in order to observe replacement effects as rapidly as possible. Hypothyroid animals without T 3 replacement show only a small uterine response to estrogen (Fig. 4, 0 time group). If hypothyroid animals are treated with both T 3 and estradiol 24 h before sacrifice, the response is enhanced (Fig. 4, 24 h time group) but is still less than that of euthyroid controls. Replacement with T 3 for 72 h (daily doses of 50 jixg/100 g BW for 3 days) yields a response 24 h after estrogen treatment which is similar to that observed in euthyroid animals (Fig. 4, 72 h time group). Treatment with this dose of T 3 for 72 h also restored estrogen-induced increases in uterine DNA
synthesis, protein, and RNA content. Note also, as previously observed (Fig. 3), that T 3 even at the high dose used in this study does not significantly affect the saline-treated control values [Fig. 4, upper panel ( )]. Since T 3 administration restores the diminished uterine response observed in hypothyroid animals, we next determined if this represented a direct effect of T 3 or if T 3 was stimulating release of pituitary GH (23) which was in turn affecting the uterus (24). In order to distinguish between these possibilities, exogenous T 3 was administered in an attempt to restore the diminished uterine response we had described previously in OVX-HX animals (17). The results of these studies are shown in Fig. 5. As previously reported (17), the uterine response to estrogen is markedly decreased in OVH-HX animals relative to OVX animals.
50
UJ I Z X
Endo • 1978 Vol 103 • No 4
60
OVX
OVX (T 3 )
25 OVX-HX (T 3 )
SALINE
E
Vol. 103, No. 4 Printed in U.S.A.
Regulation of the Uterine Response to Estrogen by Thyroid Hormone* RUSSELL M. GARDNERS JOHN L. KIRKLAND, JUDY S. IRELAND, AND GEORGE M. STANCEL^ Departments of Pharmacology and Pediatrics, The University of Texas Medical School at Houston, Houston, Texas 77025 ABSTRACT. After estradiol (E2) administration, early increases (within 4 h) in uterine wet weight and the synthesis of 2-deoxyglucose-6-phosphate from 2-deoxyglucose are similar in ovariectomized rats and in ovariectomized rats made hypothyroid by feeding a low iodine diet containing propylthiouracil. Most late uterine responses occurring 24 h after E2 treatment, however, are greatly diminished in the hypothyroid animals. The diminished responses include increases in uterine wet weight, dry weight, protein content, RNA content, and the incorporation of thymidine into uterine DNA. One response, the synthesis of 2-deoxyglucose-6-phosphate from 2-deoxyglucose is not diminished in hypothyroid rats 24 h after E2 treatment. The diminished uterine response is not due to a shift in the dose-response curve
for E2, but results from a decrease in the magnitude of the maximum uterine response. Treatment of hypothyroid rats with exogenous T,-i restores the diminished uterine response in a dose-dependent and time-dependent manner. A dose of 0.5 /xg T:1/100 g BW for 5 days restores the response completely, while 48-72 h of treatment with higher doses of T.i are required to restore the response. The effect of T.) is not mediated by the pituitary, since exogenous T.i restores diminished uterine responses in ovariectomized, hypophysectomized animals. These results suggest that thyroid hormones may have a direct effect on the uterus which regulates the responsiveness of the organ to E2. (Endocrinology 103: 1164, 1978)
T
HE OVERALL response of the rat uterus to estrogens is a complex process which seems to involve at least two major temporal phases, an early phase and a late phase (1-5). Specific responses occurring in the early phase are increases in uterine wet weight (3, 6, 7), glucose metabolism (7-9), and RNA polymerase activities (10-13), while late responses include increases in uterine dry weight (4-6), DNA synthesis (9, 14), protein (10, 15), and RNA content (10, 15). It has been suggested that these late uterine responses represent true growth of the organ, while the early responses represent a preparation for this
growth. The complete uterine response seems to be mediated by a series of interactions in which hormonal regulation may exist at multiple sites within uterine nuclei and at various times after hormone treatment (see References 5 and 16 for recent reviews). In previous work we observed that hypophysectomy could selectively diminish most late uterine responses to estrogen occurring 24 h after hormone treatment, but had little effect on uterine responses occurring within 4 h after estrogen administration (17). Therefore, we initiated a series of studies to determine which specific pituitary factor(s) was responsible for the decreased uterine response observed in hypophysectomized animals. Received October 21, 1977. Address requests for reprints to: Dr. George M. StanIn this study we have observed that thyroid eel, University of Texas, Department of Pharmacology, hormones regulate the uterine response to Health Sciences Center, P.O. Box 20708,6400 West Cullen estrogens. Most late uterine responses to esStreet, Houston, Texas 77025. * This work was supported by NIH Grant HD-08615. trogens are markedly decreased in hypothyA preliminary report of this investigation was presented roid animals, and this effect is reversed by the at the Federation of American Societies for Experimental Biology, Chicago, IL, April 1977 (FedProc 36: 368,1977). administration of exogenous T3 in a dose-def Recipient of NIH Research Fellowship Award AM- pendent and time-dependent manner. Exoge05246. Present address: Department of Biology, Rochester nous T also restores the diminished uterine 3 Institute of Technology, Rochester, New York. response to estrogen previously observed in $ Recipient of NIH Research Career Development hypophysectomized animals (17). These reAward 1 K04 HD-00099. 1164 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 28 November 2015. at 00:23 For personal use only. No other uses without permission. . All rights reserved.
THYROID REGULATION OF ESTROGEN ACTION
suits indicate that the decreased uterine response observed in hypophysectomized animals results from hypothyroidism secondary to decreased levels of TSH, and further suggest that thyroid hormones may have a direct effect on the uterus which regulates the responsiveness of this organ to estrogens. Materials and Methods Rats used for the majority of studies in this work were obtained from Texas Inbred Mouse Co., Houston, TX. All animals used in this work were ovariectomized at 22-24 days of age (approximately 50 g BW) and were allowed to recover for 1-2 days. Animals were then fed ad libitum either a diet of normal rat chow or a low iodine diet containing 0.15% propylthiouracil (PTU) obtained from Teklad, Madison, WI, and given drinking water for 30 days before use. At this time, animal weights ranged from 156-225 g in the group receiving the normal diet, and from 64-90 g in those receiving the PTU diet. Serum T 4 levels were also measured at this time by RIA of serum from randomly selected rats in both groups. These measurements were performed by Veterinary Laboratory Associates, Houston, TX. Mean serum T4 levels for the normal group were 3.46 /xg/100 ml ± 0.19 (n = 11) and for the PTU groups were 0.58 jug/100 ml ± 0.08 (n = 9). For studies with hypophysectomized animals, either ovariectomized (OVX) or ovariectomized, hypophysectomized (OVX-HX) rats were purchased from Zivic-Miller Laboratories, Allison Park, PA, and maintained as previously described (17). Before sacrifice, animals were injected ip with the doses of 17/S-estradiol (Schwarz/Mann) indicated in the text for individual experiments or with the vehicle alone (control groups). Estradiol was routinely administered in 0.5 ml 95% saline-5% EtOH, except in the dose-response studies where 0.5 ml 50% saline-50% propylene glycol was used. Animals were sacrificed by decapitation and the uteri were removed, stripped of adhering fat and mesentery, and weighed on a Cahn Electrobalance. Uterine dry weights were measured with a Mettler Analytical Balance after drying (90 C) to constant weight. Uterine DNA synthesis was measured as described by Kaye et al. (14). Uteri were incubated in Eagle's HeLa medium (Difco) for 1 h at 37 C with 0.25 juCi [3H]thymidine (Schwarz/Mann)/ml and a total thymidine concentration of 4 X 10"b M. Incubations were performed under 95% O2-5% CO2 with
1165
gentle shaking. After the incubation the uteri were homogenized, and nucleic acids and proteins were precipitated by perchloric acid (PCA; 0.25 N) at 0 C. After washing, the pellets were hydrolyzed at 90 C for 30 min in the presence of 0.4 N PCA. Aliquots of the final supernatants were then counted to dtermine [3H]thymidine incorporation. The conversion of 2-deoxyglucose to 2-deoxyglucose-6-phosphate was measured as described by Gorski and Raker (18). Uteri were incubated in Eagle's HeLa medium containing 0.5 juCi/ml 2-[l14 C]deoxyglucose (New England Nuclear) for 30 min at 37 C under an atmosphere of 95% O2-5% CO2. After homogenization and removal of PCAinsoluble material, the samples were adjusted to alkaline pH (approximately 8.0) and insoluble potassium perchlorate was removed by centrifugation. The I4C-labeled 2-deoxyglucose-6-phosphate formed was then absorbed with Bio-Rad Ag2-X8 resin in a batch procedure, the resin was washed three times with 4.0 ml distilled water, and then extraction was performed with HC1 (0.2 N) to remove the 2-deoxyglucose-6-phosphate for radioactive counting. Protein content was measured by a modification (19) of the method of Lowry et al, and RNA content was measured by the orcinol reaction (20). Where indicated in the text, L-T 3 (Sigma) was administered sc in dilute base at the dose levels and times described for individual experiments. For studies of the retention patterns of nuclear estrogen receptor complexes, animals were injected ip with 0.2 jug [3H]estradiol (New England Nuclear)/100 g BW (SA, 10-15 Ci/mmol). In parallel experiments, animals received the same dose of [3H]estradiol plus a 100-fold excess of unlabeled hormone to correct for nonspecific binding. At the times indicated in the text, animals were sacrificed, uterine nuclei were prepared and washed, and [3H]estradiol was extracted from the nuclei with 95% EtOH, as previously described (21). All values indicated in the text and figures represent specific binding. Serum GH levels were kindly measured by Dr. Max Hutchins, Department of Physiology, The University of Texas Dental School, Houston, TX, using RIA, as described previously (22).
Results The data in Fig. 1 illustrate the uterine response at various times after estrogen administration to normal and hypothyroid rats. Both groups of animals show an initial increase in wet weight at 4 h, which is compa-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 28 November 2015. at 00:23 For personal use only. No other uses without permission. . All rights reserved.
GARDNER ET
1166
Endo , 1978 Vol 103 , No 4
AL.
In addition to the data in Fig. 1, other uterine responses occurring 24 h after estrogen administration are also greatly diminished in hypothyroid animals. These results are illustrated in Table 1. Since we had previously observed that hyT —T pophysectomy did not appreciably diminish ^ T — 60 NORMAL estrogen-stimulated uterine glucose metabo/I 40 lism (17), it was next of interest to study this 2 I -o parameter in hypothyroid animals. The reE o 20 ' PTU sults of these studies are depicted in Table 2, si 0 which illustrates the effect of estrogen treatment on the synthesis of 2-deoxyglucose-6ZbU phosphate from 2-deoxyglucose 4 and 24 h 220 after treatment in normal and hypothyroid animals. Unlike the other parameters studied, i 180 2-deoxyglucose-6-phosphate synthesis is not NORMAL 140 decreased in hypothyroid animals. We next determined whether the decreased PTU 100 response observed in hypothyroid animals 24 5 h after estrogen treatment (Fig. 1) was due to a decrease in the maximum uterine response 12 24 36 0 4 TIME (Hours) or a shift in the dose-response curve for estraFIG. 1. Time course of estradiol (E2) action in normal diol. The results of studies designed to differand hypothyroid (PTU) animals. Animals were treated entiate these two possibilities are illustrated with E2 (4 jug/100 g BW) at the indicated times before in Fig. 2. This figure reveals that the uterine sacrifice. Saline-treated control values did not change response to estrogen is maximum at 0.4 jug over the time course shown (data not shown). Upper panel, Uterine wet weight values; lower panel, responses estradiol/100 g BW in both groups, and that as percentages of zero time controls. Values represent larger doses of the hormone fail to restore the uterine response in hypothyroid animals. means with the indicated SEM (n = 6-7/point). rable relative to saline-treated controls in both groups. However, at longer times, e.g. 24 h, the response is clearly diminished in the hypothyroid animals.
h ,
f'
•'•---6
^
TABLE 1. Effect of hypothyroidism on the uterine response to estradiol (E2) 24 h after treatment Treatment Parameter Uterine dry weight (mg/uterus) Normal PTU Uterine protein content (mg/uterus) Normal PTU Uterine RNA content (/xg/uterus) Normal PTU Uterine DNA synthesis (cpm [:)H]thymidine incorporated//^ DNA) Normal PTU
Response as % of saline control
Saline
E2
4.25 ± 0.27 (6) 2.74 ± 0.14 (7)
6.42 ± 0.27 (6) 2.96 ± 0.27 (7)
151 ± 6 108 ± 10
2.87 ± 0.21 (7) 2.15 ± 0.09 (7)
5.18 ± 0.37 (7) 2.82 ± 0.13 (7)
180 ± 13 131 ± 6
91.6 ± 10 (7) 92.0 ± 9.4 (7)
240 ± 27 (7) 128 ± 18 (7)
262 ± 29 139 ± 19
30.5 ± 1.9 (7) 22.0 ± 0.7 (7)
208 ± 51 (7) 39.5 ± 2.3 (7)
682 ± 168 180 ± 11
Animals were mantained on a normal diet (Normal) or a PTU-containing diet, as described in Materials and Methods. Animals were then treated with E2 (4 jug/100 g BW) or the vehicle alone (saline) 24 h before sacrifice. All values represent means ± SEM; the number of animals used for each determination is in parentheses. E2 treatment produced significant increases (P < 0.05) in all parameters for both groups of animals, except for dry weight and RNA content in the hypothyroid (PTU) animals; but increases produced by E2 treatment (response as a percentage of saline control) in the normal animals are significantly different {P < 0.005) from those produced in hypothyroid animals for all parameters.
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THYROID REGULATION OF ESTROGEN ACTION TABLE 2. Effect of estradiol (E2) treatment on 2-deoxyglucose-6-phosphate synthesis in normal and hypothyroid rats Treatment
Re-
Saline
E,
sponse as % of saline control
PTU
1132 ± 42 (4) 1074 ± 48 (5)
2028 ±246 (6) 1940 ± 9 1 (6)
179 ± 22 181 ± 9
24 H Normal PTU
714 ± 103 (5) 547 ± 36 (6)
1238 ± 147 (7) 966 ± 7 0 (7)
173 ± 21 177 ± 13
Time of sacrifice 4H Normal
Animals were fed a normal diet (Normal) or a PTUcontaining diet, as described in Materials and Methods. Either 4 or 24 h before sacrifice, animals were treated with 4 /xg/100 g BW E2 or the vehicle alone (saline). The synthesis of 2-deoxyglucose-6-phosphate from 2-deoxyglucose was then measured, as described in Materials and Methods. Values (counts per min per uterus) represent means ± SEM; the number of animals in each group is given in parentheses.
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In order to insure that the decreased long term uterine responses observed in the preceding studies were due to decreased levels of thyroid hormones, it was important to demonstrate that the observed effects could be reversed by treatment with exogenous thyroid hormone. The data in Fig. 3 thus illustrate the effect of T3 administration on the uterine response to estradiol in hypothyroid rats. These results illustrate several salient features. First, T 3 alone does not elicit any uterine response (Fig. 3, upper panel: saline controls). Second, T 3 administration to hypothyroid animals restores the ability of the uterus to respond to estradiol 24 h after treatment. A dose of 0.5 jug T3/IOO g BW restores the response completely, and a dose of 0.05 jug T 3 is approximately half-maximal. The dose-response curves for T 3 restoration of estrogen-induced increases in uterine protein and RNA content
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FIG. 2. Estradiol (E2) dose-response curves for normal and hypothyroid (PTU) animals. Animals were treated with the indicated doses of E2 24 h before sacrifice. Upper panel, Uterine wet weight values; lower panel, responses plotted as percentages of control (animals not receiving E2). Values represent means with the indicated SEM (n = 5-8/point).
Dose-response studies for increases in other uterine parameters, e.g. uterine protein and RNA content, 24 h after hormone treatment were similar to those observed in Fig. 2.
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FIG. 3. Effect of T:J on the uterine response to estradiol (E2) in hypothyroid animals. Hypothyroid animals were treated daily with the indicated total dose of T3 for 5 days before sacrifice. Twenty-four hours before sacrifice animals at each dose level of T3 were treated with either saline {upper panel, ) or 4 jug/100 g BW E 2 (upper panel, ). Lower panel, Responses as percentages of control animals not receiving T3. The open square near the ordinate (lower panel) represents the response of normal animals to E2 relative to appropriate salinetreated controls. Values represent means with the indicated SEM (n = 6-7/point).
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24 h after treatment are also similar. The full restoration of the uterine response to estrogen requires 2-3 days, as illustrated in Fig. 4. For these studies a large dose of T3 (50 jug/100 g BW) was used in order to observe replacement effects as rapidly as possible. Hypothyroid animals without T 3 replacement show only a small uterine response to estrogen (Fig. 4, 0 time group). If hypothyroid animals are treated with both T 3 and estradiol 24 h before sacrifice, the response is enhanced (Fig. 4, 24 h time group) but is still less than that of euthyroid controls. Replacement with T 3 for 72 h (daily doses of 50 jixg/100 g BW for 3 days) yields a response 24 h after estrogen treatment which is similar to that observed in euthyroid animals (Fig. 4, 72 h time group). Treatment with this dose of T 3 for 72 h also restored estrogen-induced increases in uterine DNA
synthesis, protein, and RNA content. Note also, as previously observed (Fig. 3), that T 3 even at the high dose used in this study does not significantly affect the saline-treated control values [Fig. 4, upper panel ( )]. Since T 3 administration restores the diminished uterine response observed in hypothyroid animals, we next determined if this represented a direct effect of T 3 or if T 3 was stimulating release of pituitary GH (23) which was in turn affecting the uterus (24). In order to distinguish between these possibilities, exogenous T 3 was administered in an attempt to restore the diminished uterine response we had described previously in OVX-HX animals (17). The results of these studies are shown in Fig. 5. As previously reported (17), the uterine response to estrogen is markedly decreased in OVH-HX animals relative to OVX animals.
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