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Synergistic effect of estrogen and androgen on induction of uterine thymidine kinase activities in immature rats
Btoch,mica et Biophystca Acta, 755 (1983) 307-309
307
Elsevier Biomedical Press
BBA Report BBA 20033
SYNERGISTIC EFFECT OF ESTROGEN AND ANDROGEN ON INDUCTION OF UTERINE THYMIDINE KINASE ACTIVITIES IN IMMATURE RATS N O R I K O Y A M A D A , S H I N O B U S A K A M O T O and RYOHEI O K A M O T O
Department of Endocrmology, Medical Research Institute, Tokyo Med*cal and Dental Umversity, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113 (Japan) (Received August 30th, 1982)
Key words: Enzyme induction; Estrogen; Androgen; Thymtdme kmase; (Rat uterus)
Estradiol-17fl induced a significant increase in the uterine thymidine kinase activity with a characteristic isozyme pattern 30 h after injection into immature rats. Testosterone propionate also revealed a similar increase. Following combined injection of estradioi-17fl and testosterone propionate, the overall and separate isozyme activities of thymidine kinase increased to nearly the total amount of those when each hormone was injected separately.
Estrogen induces uterine DNA replication after binding to specific receptors [1,2]. Our previous report using immature rat uterus indicated that estrogen induced remarkable increases in activities of overall thymidine kinase and one of the thymidine kinase isozymes separated by DEAE-cellulose column chromatography [3]. Androgen receptors are found in rat uterus [4-6] and androgen stimulates uterine cell proliferation [7,8] like estrogen, but the differences in the mechanism of action between estrogen and androgen are still unknown. In the present study, the interaction of uterine thymidine kinase activity with estrogen and/or androgen was investigated in immature rats. Estradiol-17fl (I.0 #g/100 g body weight, Merck, Darmstadt) and/or testosterone propionate (1.0 mg/100 g body weight, Merck) were injected intramuscularly into SD-strain immature female rats, 21 days-of-age. Control animals were treated with the vehicle in a similar manner. All rats were killed by cervical dislocation 30 h after the last injection. As previously reported [3], uterine thymidine kinase activity was assayed by the method of Taylor et al. [9], and uterine thymidine kinase isozymes were separated by DEAE-cel0304-4165/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
lulose column chromatography. As shown in Table I, testosterone induced a remarkable increase, the same as that induced by 17fl-estradiol, in uterine thymidine kinase activity in immature rats. Thymidine kinase activity began to increase 18 h after testosterone administration and peaked after 30 h followed by a decline to the TABLE I EFFECTS O F E S T R O G E N A N D T E S T O S T E R O N E ON T H E T H Y M I D I N E K I N A S E ACTIVITY IN R A T U T E R U S Uteri from 17fl-estrad]ol- and testosterone-treated rats w e r e homogenized with 10 volumes of 50 m M Tris-HCl (pH 7.5) buffer, 1 m M EDTA, 5 m M mercaptoethanol, and then centrifuged at 1 0 5 0 0 0 × g for l h. The supernatant was usecl for enzyme assay. Mean ± S.D. ( n = 5). Thymidine kinase activity (dpm- 10- 4 / m g protein per nun) Control 17~-Estradioi Testosterone 17~O-Estradiol + testosterone
2.14+ 0,51 102.0 + 60.0 117.0 + 43.0 174.0 + 87.0 a
" P < 0.02 for estradiol, testosterone.
308
basal level after 72 h. This time course of thymidine kinase activity induced by testosterone was similar to that induced by 17fl-estradiol [3]. Combined administration of 17/3-estradiol and testosterone induced a greater increase in the activity than that induced by 17/3-estradiol or testosterone alone. As reported previously, uterine thymidine kinase activity in 17/3-estradiol-treated immature rats was separated into three peaks (peaks A, B and C) by DEAE-cellulose column chromatography [3] (Fig. 1). Peak A was found to have different enzymatic properties from peaks B and C, and might be involved in D N A replication
[31. Similarities in the elution patterns were found in 17/3-estradiol- and testosterone-treated uteri. There were no differences among the 3 peaks induced by 17/3-estradiol or testosterone with regard to the effects of dTTP and the mobilities in polyacrylamide gel electrophoresis. Combined administration of 17/3-estradiol and testosterone induced a higher activity in each isozyme than that induced by 17/3-estradiol or testosterone alone.
c -
_ _ J
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23
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80 r •
TABLE II TOTAL ACTIVITIES OF UTERINE T H Y M I D I N E KINASE ISOZYMES SEPARATED BY DEAE-CELLULOSE COLUMN C H R O M A T O G R A P H Y Acttvity of each thymldlne kunase isoz_'~me was estimated from c h r o m a t o g r a m s s h o w n in Fig I
Thymldme kmase aCtlVlV, (cpm-10 5/mm)
17fl-EstradLol Testosterone 17,8-Estrad,ol + testosterone
A
B
C
153 92 210
42 32 71
113 63 208
Table II shows total activity of each thymidine kinase isozyme shown in Fig. 1. Combined administration of 17/3-estradiol and testosterone increased each thymidine kinase isozyme activity, which was approximately the sum of those induced by 17/3estradiol and testosterone alone. It seems that 17fl-estradiol plus testosterone also had a synergistic effect on the activity of each thymtdine kinase isozyme. To clarify this synergistic effect of 17/3-estradiol and testosterone on uterine thymidine kinase, experiments of repeated injection with 17/3-estradiol or testosterone were conducted. The second injec-
10
TABLE III
E
EFFECTS OF REPEATED INJECTION WITH ESTROGEN OR TESTOSTERONE ON THE T H Y M I D I N E KINASE ACTIVITY IN RAT UTERUS
5
10
20
10
2O
frachon
Fig. 1. Effects of estrogen and testosterone on the thym~dnne k m a s e lsozyme actiwties in rat uterus. Each 1 g of uteri separated 30 h after h o r m o n e injectton were h o m o g e n i z e d with 3 v o l u m e s of 50 mM tns-HCI (pH 7.5) buffer/1 mM E D T A / 5 mM mercaptoethanol. The h o m o g e n a t e was centrifuged at 15000::
Cytosol of uteri 60 h after first h o r m o n e rejection were used as e n z y m e source The second injection was performed after an interval of 30 h. H o r m o n e rejection Ftrst
Second
-17fl-Estradnol 17fl-Estradlol 17,8-Estradlol
--17,8-Estradlol Testosterone
" P < 0.01, mean_+S D. (n = 10)
T h y m i d m e kma~e activity (dpm-10 4/mg protein per nun) 6.5_+ 13 42.2 _+ 16 3 40 4 + 12.7 75.6 + 15 6 ~
309 tion was performed after an interval of 30 h and the thymidine kinase activity was assayed 30 h after the second injection (Table III). Although the second injection of 17fl-estradiol scarcely raised the uterine thymidine kinase activity in 17/3estradiol-primed immature rats, when the second injection was testosterone instead of 17fl-estradiol, a significant increase in the activity was brought about. It has been reported that a large dose of androgen is able to bind to the estrogen receptor in the uterus [10]. However according to recent other reports, testosterone and 5 a - d i h y d r o testosterone have strong affinities for uterine androgen receptors [6], and testosterone dose not bind to the estrogen receptor in vivo [5,11,13] without affecting the plasma estrogen level [12]. These reports suggest that the effect of testosterone on the uterine thymidine kinase activity in the present study was induced with no relation to the estrogen receptors. The present results indicate that estrogen or androgen independently raise the uterine thymidine kinase activity in immature rats, although the mechanisms are unknown. There are two possibilities. One is that androgen stimulates a different kind of cell in the uterine tissue from estrogen. Another possibility is that b o t h estrogen and androgen act on the same kind of cell in the uterus. In 1954, Huggins et al. [7] reported that estrogen and androgen have the same target cells in the vagina of rats. However, since then, few studies on uterine target cells for androgen have been carried out. In 1981, Hager et al. [14] showed that in the chick oviduct, dexametha-
son shows synergistic action with estrogen in the induction of ovalbumin and conalbumin by binding to its receptor which is distinct from the estrogen receptor. If estrogen and androgen might act on the same cells in uterus, it can be considered that uterine thymidine kinase activity is controlled by different system which interact with the specific steroid hormone. Further studies are underwayed on the above two possibilities,
References 1 Tlce, L.W. (1961) Anat. Rec. 139, 515-523 2 Jensen, E.V. and de Sombre, E.R. (1972) Annu. Rev. Biochem. 41,203-230 3 Yamada, N., Sakamoto, S., Sawasaki, Y., Nakajima, H. and Okamoto, R. (1980) Biochim. Biophys. Acta 629, 61-68 4 Giannopoulos, G. (1971) Biochem. Biophys. Res. Commun. 44, 943-951 5 Poortman, J., Prenen, J.A.C., Schwarz, F. and Thijssen, J.H.H. (1975) J. Clin. Endocrinol. Metab. 40, 373-379 6 Rochefort, H. and Lignon, F. (1974) Eur. J. Biochem. 48, 503-512 7 Huggins, C., Jensen, E.V. and Cleveland, A.S. (1954) J. Exp. Med. 100, 225-240 8 Lerner, L.J., Hilt', R., Tuekheimer, A.R., Michel, I. and Engel, S.L. (1966) Endocrinology 78, I 11-124 9 Taylor, A.T., Stafford, M.A. and Jones, O.W. (1972) J. Biol. Chem. 247, 1930-1935 10 Garcla, M. and Rochefort, H. (1979) Endocrinology 104, 1979-1804 11 Schmldt. W.N., Sadler, M.A. and Katzenellenbogen, B.S. (1976) Endocrinology 98, 702-716 12 Udagawa, H. (1980) Folia Endocrinol. Jap. 56, 1172-1181 13 Watson, G.H., Korach, K.S. and Muldoon, T.G. (1977) Endocrinology 101, 1733-1743 14 Hager, L.J., McKnight, G.S. and Palmiter, R.D. (1980) J. Biol. Chem. 255, 7796-7800
307
Elsevier Biomedical Press
BBA Report BBA 20033
SYNERGISTIC EFFECT OF ESTROGEN AND ANDROGEN ON INDUCTION OF UTERINE THYMIDINE KINASE ACTIVITIES IN IMMATURE RATS N O R I K O Y A M A D A , S H I N O B U S A K A M O T O and RYOHEI O K A M O T O
Department of Endocrmology, Medical Research Institute, Tokyo Med*cal and Dental Umversity, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113 (Japan) (Received August 30th, 1982)
Key words: Enzyme induction; Estrogen; Androgen; Thymtdme kmase; (Rat uterus)
Estradiol-17fl induced a significant increase in the uterine thymidine kinase activity with a characteristic isozyme pattern 30 h after injection into immature rats. Testosterone propionate also revealed a similar increase. Following combined injection of estradioi-17fl and testosterone propionate, the overall and separate isozyme activities of thymidine kinase increased to nearly the total amount of those when each hormone was injected separately.
Estrogen induces uterine DNA replication after binding to specific receptors [1,2]. Our previous report using immature rat uterus indicated that estrogen induced remarkable increases in activities of overall thymidine kinase and one of the thymidine kinase isozymes separated by DEAE-cellulose column chromatography [3]. Androgen receptors are found in rat uterus [4-6] and androgen stimulates uterine cell proliferation [7,8] like estrogen, but the differences in the mechanism of action between estrogen and androgen are still unknown. In the present study, the interaction of uterine thymidine kinase activity with estrogen and/or androgen was investigated in immature rats. Estradiol-17fl (I.0 #g/100 g body weight, Merck, Darmstadt) and/or testosterone propionate (1.0 mg/100 g body weight, Merck) were injected intramuscularly into SD-strain immature female rats, 21 days-of-age. Control animals were treated with the vehicle in a similar manner. All rats were killed by cervical dislocation 30 h after the last injection. As previously reported [3], uterine thymidine kinase activity was assayed by the method of Taylor et al. [9], and uterine thymidine kinase isozymes were separated by DEAE-cel0304-4165/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
lulose column chromatography. As shown in Table I, testosterone induced a remarkable increase, the same as that induced by 17fl-estradiol, in uterine thymidine kinase activity in immature rats. Thymidine kinase activity began to increase 18 h after testosterone administration and peaked after 30 h followed by a decline to the TABLE I EFFECTS O F E S T R O G E N A N D T E S T O S T E R O N E ON T H E T H Y M I D I N E K I N A S E ACTIVITY IN R A T U T E R U S Uteri from 17fl-estrad]ol- and testosterone-treated rats w e r e homogenized with 10 volumes of 50 m M Tris-HCl (pH 7.5) buffer, 1 m M EDTA, 5 m M mercaptoethanol, and then centrifuged at 1 0 5 0 0 0 × g for l h. The supernatant was usecl for enzyme assay. Mean ± S.D. ( n = 5). Thymidine kinase activity (dpm- 10- 4 / m g protein per nun) Control 17~-Estradioi Testosterone 17~O-Estradiol + testosterone
2.14+ 0,51 102.0 + 60.0 117.0 + 43.0 174.0 + 87.0 a
" P < 0.02 for estradiol, testosterone.
308
basal level after 72 h. This time course of thymidine kinase activity induced by testosterone was similar to that induced by 17fl-estradiol [3]. Combined administration of 17/3-estradiol and testosterone induced a greater increase in the activity than that induced by 17/3-estradiol or testosterone alone. As reported previously, uterine thymidine kinase activity in 17/3-estradiol-treated immature rats was separated into three peaks (peaks A, B and C) by DEAE-cellulose column chromatography [3] (Fig. 1). Peak A was found to have different enzymatic properties from peaks B and C, and might be involved in D N A replication
[31. Similarities in the elution patterns were found in 17/3-estradiol- and testosterone-treated uteri. There were no differences among the 3 peaks induced by 17/3-estradiol or testosterone with regard to the effects of dTTP and the mobilities in polyacrylamide gel electrophoresis. Combined administration of 17/3-estradiol and testosterone induced a higher activity in each isozyme than that induced by 17/3-estradiol or testosterone alone.
c -
_ _ J
-
23
__J
5
80 r •
TABLE II TOTAL ACTIVITIES OF UTERINE T H Y M I D I N E KINASE ISOZYMES SEPARATED BY DEAE-CELLULOSE COLUMN C H R O M A T O G R A P H Y Acttvity of each thymldlne kunase isoz_'~me was estimated from c h r o m a t o g r a m s s h o w n in Fig I
Thymldme kmase aCtlVlV, (cpm-10 5/mm)
17fl-EstradLol Testosterone 17,8-Estrad,ol + testosterone
A
B
C
153 92 210
42 32 71
113 63 208
Table II shows total activity of each thymidine kinase isozyme shown in Fig. 1. Combined administration of 17/3-estradiol and testosterone increased each thymidine kinase isozyme activity, which was approximately the sum of those induced by 17/3estradiol and testosterone alone. It seems that 17fl-estradiol plus testosterone also had a synergistic effect on the activity of each thymtdine kinase isozyme. To clarify this synergistic effect of 17/3-estradiol and testosterone on uterine thymidine kinase, experiments of repeated injection with 17/3-estradiol or testosterone were conducted. The second injec-
10
TABLE III
E
EFFECTS OF REPEATED INJECTION WITH ESTROGEN OR TESTOSTERONE ON THE T H Y M I D I N E KINASE ACTIVITY IN RAT UTERUS
5
10
20
10
2O
frachon
Fig. 1. Effects of estrogen and testosterone on the thym~dnne k m a s e lsozyme actiwties in rat uterus. Each 1 g of uteri separated 30 h after h o r m o n e injectton were h o m o g e n i z e d with 3 v o l u m e s of 50 mM tns-HCI (pH 7.5) buffer/1 mM E D T A / 5 mM mercaptoethanol. The h o m o g e n a t e was centrifuged at 15000::
Cytosol of uteri 60 h after first h o r m o n e rejection were used as e n z y m e source The second injection was performed after an interval of 30 h. H o r m o n e rejection Ftrst
Second
-17fl-Estradnol 17fl-Estradlol 17,8-Estradlol
--17,8-Estradlol Testosterone
" P < 0.01, mean_+S D. (n = 10)
T h y m i d m e kma~e activity (dpm-10 4/mg protein per nun) 6.5_+ 13 42.2 _+ 16 3 40 4 + 12.7 75.6 + 15 6 ~
309 tion was performed after an interval of 30 h and the thymidine kinase activity was assayed 30 h after the second injection (Table III). Although the second injection of 17fl-estradiol scarcely raised the uterine thymidine kinase activity in 17/3estradiol-primed immature rats, when the second injection was testosterone instead of 17fl-estradiol, a significant increase in the activity was brought about. It has been reported that a large dose of androgen is able to bind to the estrogen receptor in the uterus [10]. However according to recent other reports, testosterone and 5 a - d i h y d r o testosterone have strong affinities for uterine androgen receptors [6], and testosterone dose not bind to the estrogen receptor in vivo [5,11,13] without affecting the plasma estrogen level [12]. These reports suggest that the effect of testosterone on the uterine thymidine kinase activity in the present study was induced with no relation to the estrogen receptors. The present results indicate that estrogen or androgen independently raise the uterine thymidine kinase activity in immature rats, although the mechanisms are unknown. There are two possibilities. One is that androgen stimulates a different kind of cell in the uterine tissue from estrogen. Another possibility is that b o t h estrogen and androgen act on the same kind of cell in the uterus. In 1954, Huggins et al. [7] reported that estrogen and androgen have the same target cells in the vagina of rats. However, since then, few studies on uterine target cells for androgen have been carried out. In 1981, Hager et al. [14] showed that in the chick oviduct, dexametha-
son shows synergistic action with estrogen in the induction of ovalbumin and conalbumin by binding to its receptor which is distinct from the estrogen receptor. If estrogen and androgen might act on the same cells in uterus, it can be considered that uterine thymidine kinase activity is controlled by different system which interact with the specific steroid hormone. Further studies are underwayed on the above two possibilities,
References 1 Tlce, L.W. (1961) Anat. Rec. 139, 515-523 2 Jensen, E.V. and de Sombre, E.R. (1972) Annu. Rev. Biochem. 41,203-230 3 Yamada, N., Sakamoto, S., Sawasaki, Y., Nakajima, H. and Okamoto, R. (1980) Biochim. Biophys. Acta 629, 61-68 4 Giannopoulos, G. (1971) Biochem. Biophys. Res. Commun. 44, 943-951 5 Poortman, J., Prenen, J.A.C., Schwarz, F. and Thijssen, J.H.H. (1975) J. Clin. Endocrinol. Metab. 40, 373-379 6 Rochefort, H. and Lignon, F. (1974) Eur. J. Biochem. 48, 503-512 7 Huggins, C., Jensen, E.V. and Cleveland, A.S. (1954) J. Exp. Med. 100, 225-240 8 Lerner, L.J., Hilt', R., Tuekheimer, A.R., Michel, I. and Engel, S.L. (1966) Endocrinology 78, I 11-124 9 Taylor, A.T., Stafford, M.A. and Jones, O.W. (1972) J. Biol. Chem. 247, 1930-1935 10 Garcla, M. and Rochefort, H. (1979) Endocrinology 104, 1979-1804 11 Schmldt. W.N., Sadler, M.A. and Katzenellenbogen, B.S. (1976) Endocrinology 98, 702-716 12 Udagawa, H. (1980) Folia Endocrinol. Jap. 56, 1172-1181 13 Watson, G.H., Korach, K.S. and Muldoon, T.G. (1977) Endocrinology 101, 1733-1743 14 Hager, L.J., McKnight, G.S. and Palmiter, R.D. (1980) J. Biol. Chem. 255, 7796-7800