- Email: [email protected]
Differential c-jun gene expression with tonically administered steroids in rat ovary and uterus
Shelley et al.
24. 25. 26. 27. 28.
May 1994
Am J Obstet Gynecol
racy and confirmation by gas chromatography/mass spectrometry. j Forensic Sci 1989;34:32-45. Mittleman RE, Cofino jC, Hearn WL. Tissue distribution of cocaine in a pregnant woman. j Forensic Sci 1989;34: 481-6. Fox H, Faulk W. The placenta as an experimental animal. Clin Endocrinol Metab 1981;10:57-72. Fox H. Placenta as a model for organ aging. In: Beaconsfield P, Villee G, eds. Placenta-a neglected experimental animal. Oxford: Pergamon Press, 1979:351-85. Hytten F, Chamberlain G. Clinical physiology in obstetrics. Oxford: Blackwell Scientific Publishers, 1980. Ng W, Miller RK. Transport of nutrients in the early
human placenta: amino acid, creatinine, vitamin B 12 • Trophoblast Res 1983;1:121-34. 29. Dancis j, Levitz M, Katz D, et al. Transfer and metabolism of retinol by the perfused human placenta. Pediatr Res 1992;32:195-9. 30. Fortunato Sj, Bawdon RE, Swan KF, Bryant EC, Sobhi S. Transfer of Timentin (ticarcillin and clavulanic acid) across the in vitro perfused human placenta: comparison with other agents. AM j OBSTET GYNECOL 1992;167:159599. 31. Ahmed MS, Zhou DE, Maulik D, Eldefrawi ME. Characterization of a cocaine binding protein in human placenta. Life Sci 1990;46:553-61.
Differential c-jun gene expression with tonically administered steroids in rat ovary and uterus Martha E. Shelley, MD, Amjad Hossain, PhD, Paul G. McDonough, MD, and Iqbal Khan, PhD
Augusta, Georgia OBJECTIVE: The purpose of this study was to evaluate the induction of the early regulatory gene c-jun in response to tonic exposure to estradiol and progesterone in rat ovary, uterus, and adrenal tissues. STUDY DESIGN: Pellets containing estradiol-17j3, progesterone, and estradiol-17j3 plus progesterone were placed subcutaneously in immature female Sprague-Dawley rats (N = 24). The ovary, uterus, and the adrenal were evaluated for c-jun expression by Northern analysis at 24 and 48 hours. RESULTS: The c-jun messenger ribonucleic acid expression in the ovary and adrenal gland was inhibited with high, non physiologic doses of estradiol in progesterone and was induced with physiologic levels of estradiol. Physiologic levels of progesterone do not appear to influence the expression of c-jun in the ovary or adrenal gland. Uterine c-jun expression to estradiol and progesterone is generally the opposite of that observed in the ovary. CONCLUSION: These findings suggest that there is both tissue and dose specificity of c-jun gene expression in steroidogenic and steroid-responsive tissues when steroid hormones are tonically administered. (AM J OSSTET GVNECOL 1994;170:1410-5.)
Key words: Protooncogene, cjun, estradiol, progesterone The interplay of the two steroid hormones estrogen and progesterone and the growth and differentiation of different tissues in the reproductive system have been the topic of investigation over a long period of time. The physiologic role of steroids in the reproductive process is now fairly well described. I, 2 However, the molecular mechanisms by which steroid hormones and From the Department oj Obstetrics and Gynecology, Medical College of Georgza. Recezved for publicatzon June 8, 1993; reVised August 11, 1993; accepted November 12, 1993. Reprint requests: Iqbal Khan, PhD, Reproductive Endocrinology Sectzon, Cj-134, Department of ObstetriCs and Gynecology, Medlcal College of Georgza, Augusta, GA 30912. Copyright © 1994 by Mosby-Year Book, Inc.
0002-9378/94 $3.00 + 0
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receptors interact to regulate genes participating in the growth or differentiation of reproductive tissues is currently under scrutiny.'·6 Recently the nuclear protooncogenes jun and fos, both components of the AP-l transcription factor, have been demonstrated to play a role in cell proliferation and differentiation and are good candidates for mediators of steroid-induced growth and differentiation. 7 '9 Some studies implicate c-jun as an "early response gene" in the cascade of steroid action on the target tissue. 10, II To date, the response of these protooncogenes to steroid hormones has been evaluated only at the physiologic level of these hormones and at short exposure times. However, in some situations the biologic system may be loaded with steroids for protracted periods far in excess of physio-
Shelley et al.
Volume 170, Number 5, Part 1 Am J Obstet Gynecol
logic levels. For example, ovulation induction in assisted reproductive techniques and menopausal hormonal replacement therapy may expose the body to steroid levels much higher than that produced endogenously.12. 13 Although the physiologic consequences of sudden administration of steroids in the body have been under investigation, the molecular consequences of such steroidal influence is still under scrutiny. In this study we attempted to evaluate the response of one of the early steroid response genes, c-jun, to tonically administered steroids (estradiol, progesterone, or both) in the rat model. Our results show that externally introduced steroids, particularly estradiol, up-regulate steroid metabolism and that the response of protooncogenes c-jun correlates with the (I) type of steroid hormone, (2) circulating level of steroid hormone, and (3) type of tissue. Material and methods
Animals and treatment. Twenty-four immature (29 days old) Sprague-Dawley rats were divided into 24and 48-hour treatment groups. These groups were further subdivided into control, estradiol-l 713 only, progesterone only, and estradiol-1713 plus progesterone subgroups, each containing three animals. With the animals under ether anesthesia 2 cm silicone elastomer pellets containing crystalline estradiol-l 713, progesterone, or both estradiol-1713 and progesterone were placed subcutaneously. The control animals underwent the surgical procedure, but no pellets were placed. At 24 and 48 hours after placement of the pellets the animals were killed, and the ovaries. uterus, and adrenal glands were removed. The tissue was placed immediately into liquid nitrogen and stored at - 80° C until ribonucleic acid (RNA) extraction was performed. RNA isolation and analysis. Total RNA was isolated from the ovaries, uterus, and adrenal glands by the RNAzol method (CinnaiBiotecx). The RNA was quantitated and monitored for purity by evaluating the 260: 280 nm absorption. For the analysis 30 /-Lg of total RNA was fractionated by electrophoresis through a 1.0% agarose-2.2 mol!L formaldehyde gel and transferred to nylon membranes. The nylon membranes were then prehybridized for 1 hour at 65° C in hybridization buffer containing complementary deoxyribonucleic acid probes labeled with phosphorus 32-deoxycytidine triphosphate (3000 Cilmmol) with an oligolabeling kit (Pharmacia, Uppsala, Sweden). The probes used for hybridization were murine c-jun complementary deoxyribonucleic acid and chick-actin complementary deoxyribonucleic acid (used for an internal standard) from American Tissue Culture Collection. After hybridization the membranes were washed under high-stringency conditions and exposed to Kodak X-AR film (Eastman Kodak, Rochester. N.Y.) for 5 days at - 80° C.
1411
The blots were then analyzed by a Shimadzu CS9000 densitometer, and the changes in experimental c-.Jun messenger RNA (mRNA) levels were compared with those in control animals. Hormonal assays. Before death, venous blood was drawn from each animal and centrifuged at 1500 revolutions/min for 10 minutes to separate serum. The serum was stored at - 20° C. The progesterone and rabbit antiprogesterone antibody were provided courtesy of Dr. T. Mills, Augusta, Georgia. The interassay and intraassay variation was 0.5 and 1.0 ng/m!, respectively. The estradiol level in the serum was measured with a standard estradiol assay kit from Diagnostic Products (Los Angeles). The interassay and intraassay variation kit was 1.5 and 5.0 pg/ml, respectively. To confirm a constant release of steroid hormone from the silicone elastomer pellets, the in vitro release pattern of the silicone elastomer pellets was obtained by placing the pellets in 2 ml of phosphate-buffered saline solution for 24, 48, and 72 hours. The progesterone and estradiol levels in the phosphate-buffered saline solution were then measured. Results
Circulating level of steroids. The circulating levels of estradiol and progesterone in rats treated with synthetic steroids subcutaneously administered in 2 cm silicone elastomer pellets is shown in Table I. Serum estradiol levels in estradiol-only treated rats and rats treated with both estradiol and progesterone show an identical pattern. In both groups a marked difference in estradiol level is seen between the 24-hour and 48-hour time periods. By 24 hours the serum estradiol level was elevated fifteenfold compared with the control level but returned toward the control level by 48 hours. The marked difference in estradiol levels between the 24- and 48-hour time periods suggests an up-regulation of estradiol metabolism, because the in vitro release pattern of the silicone elastomer pellets was linear over 72 hours (Fig. 1). In contrast, the serum progesterone level did not reflect any marked difference between the treatment groups (estradiol-l 713 vs progesterone vs estradiol-1713 plus progesterone) over the two time periods (24 vs 48 hours). c-jun mRNA expression. The response of protoonco gene c-jun gene expression in ovary. adrenal gland, and uterus to the synthetic steroid treatment is shown in Figs. 2, 3, and 4, respectively. As seen in Figs. 2 and 5, all three treatment groups of the ovary exhibit a common pattern of expression of c-jun between the 24-hour and 48-hour time periods. The c-jun expression appears to be inhibited in the first 24 hours of exposure to exogenous progesterone and combined estradiol-1713 and progesterone treatment. This inhibition of c-jun occurs with the high level of circulating
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May 1994 Am J Obstet Gynecol
Shelley et al.
75 --0--
S
progesterone
- - estradiol
50
-----
o~~~-----+----------+---------~
o
24
48
72
Hours Fig. 1. Measurement of concentration of steroid hormone released from 2 em silicone elastomer pellet after placement in 2 ml of phosphate-buffered saline solution for 24, 48, and 72 hours. Estradiol (trzangles) is expressed as picograms per milliliter, and progesterone (square~) is expressed as nanograms per milliliter at 24, 48, and 72 hours after placement of 2 em silicone elastomer pellet in 2 ml of phosphate-buffered saline solution.
Table I. Circulating levels of estradiol and progesterone in rats treated for 24 and 48 hours with 2 cm silicone elastomer pellets containing crystalline estradiol-1713, progesterone, or both &tradlOl (pg/ml, mean ± SEM) Treatment
Control Estradiol Progesterone Estradiol plus progesterone
24 hr
186 4214 38 3599
± ± ± ±
1.5 4.3 4.1 3.1
estradiol evident at 24 hours (Table I, Fig. 2) in each treatment group. On the other hand, induction of c-jun expression in the 48-hour treatment period is seen when the circulating serum level of steroids is closer to physiologic levels (Table I, Fig. 2). By 48 hours c-jun gene expression was increased 300% in both estradiol1713 and estradiol-1713 plus progesterone treatment groups (Fig. 2). There was no increase or inhibition observed in the ovary in the presence of progesterone, suggesting no synergistic effect of progesterone (Fig. 2). Interestingly, a similar pattern of response to that seen in the ovary was evident in the adrenal, another steroidogenic tissue (Figs. 2 and 3). However, in a steroidresponsive tissue (e.g., uterus) the cjun gene appears to behave differently compared with the response in the ovary and adrenal gland (Figs. 2, 3, and 4). In the uterus there is no sign of c-jun induction; instead suppression is evident in the 48-hour treatment period for all three treatment groups (Fig. 4).
Comment Our study indicates that protracted exposure to steroid hormones results in a differential response of cjun
I
48 hr
182 298 116 308
± 1.7 ± 1.6 ± 4.2 ± 13
Progesterone (ng/ml, mean ± SEM) 24 hr
3.1 2.4 5.4 4.1
± ± ± ±
1.5 3.2 16.4 7.3
I
48 hr
3.4 6.6 3.7 4.6
± ± ± ±
5.3 1.3 6.8 13
expression in steroid-responsive and steroidogenic tissues. This is the first such report of which we are aware that evaluates cjun expression after tonic administration of steroid hormones. The steroid delivery system that uses silicone elastomer pellets for continuous release is well documented in the literature. 14. 15 The pattern of circulating hormone levels observed in our study is consistent with those reported by other investigators. In our study a marked difference in estradiol levels was seen between 24- and 48-hour treatment periods when circulating levels of steroids were analyzed (Table I). Possible up-regulation of steroid metabolism might be responsible for this wide difference in estradiol concentrations between these two time points. Analysis of either the metabolites of estradiol or its clearance from the system will be required to substantiate this speculation. The metabolic clearance of estradiol involves hepatic conjugation, conversion to estrone or estriol, and renal excretion. In previously reported studies factors that influence the rate of clearance of estradiol include dosage, hepatic blood flow, and time of infusion. 16. 17 With dosages of estradiol far in excess of physiologic levels, such as those seen during the first 24 hours of this study, the metabolic
Shelley et al.
Volume 170, Number 5, Part 1 Am J Obstet Gynecol
1413
300 •
250
-- 200
"0
24hrs
048 hrs
'~
0
u
......0 -<
z
~
E
~
150 100 50 0 Estradiol
Estradiol
Progesterone
+ Progesterone
Fig. 2. Graphic representation of cjun mRNA expression in ovary of rats after treatment for 24 and 48 hours with 2 cm silicone elastomer pellets containing crystalline estradiol-17[3, progesterone, or both. Results shown are percent mRNA level of expression of control levels.
200
--
"0 '-
•
24hrs
o 48hrs
150
~
0
u
...... 0
z-<
100
~
E
~
50 0
Estradiol
Progesterone
Estradiol
+ Progesterone
Fig. 3. Graphic representation of c-jun mRNA expression in adrenal gland of rats after treatment for 24 and 48 hours with 2 cm silicone elastomer pellets containing crystalline estradlOl-17[3, progesterone. or both. Results shown are percent mRNA level of expression of control levels.
clearance rate of estradiol has been reported to be significantly increased. IR Also, the clearance rate of estradiol does not appear to reach a steady state for at least 12 hours, in contrast to the 2 hours reported for progesterone. 19,20 The circulating values obtained in our study are consistent with the observations. On the basis of these findings, it can be speculated that a similar pattern of up-regulation may be evident in the case of hormone replacement therapy in the human. However, it is important to note that this study is based on immature rats; thus it remains open to assess whether a similar case is seen in animals that are of varying ages, particularly old age. The important observation of our experiment is that there were differential responses of the cjun gene to
tonically administered steroids. First of all, there was a differential response of the cjun gene to estradiol and progesterone treatment. The influence of progesterone on cjun is insignificant compared with that of estradiol. Poor sensitivity of cjun to progesterone treatment may in part explain this difference. In contrast, the response of cjun to treatment with estradiol was found to be dose dependent and variable among the tissues. The unusually high estradiol level (pharmacologic dose) at the 24-hour treatment period correlates with suppression of c-.Jun expression in all three tissue samples. It appears that when the system was saturated with high levels of estradiol not only complete desensitization of the tissues occurs, but the cellular physiology was also negatively affected. As mentioned earlier, the C-.JUIl
1414 Shelley et al.
May 1994 Am J Ob,tet Gynecol
150 .
as....
....>
c 0
u
24 hrs
048 hrs
100
a
<:
:z 0:::
E
50
~
0
Estradiol
Estradiol +
Progesterone
Progesterone Fig. 4. Graphic representation of c-jun mRNA expression in uterus of rats after treatment for 24 and 48 hours with 2 cm silicone elastomer pellets containing crystalline estradlOl-17J3, progesterone, or both. Results ~hown are percent mRNA level of expression of control levels.
2
3
4
5
6
7
8
c-jun
2.6 kb
actin
2.0 kb
24 hours
48 hours
Fig. 5. Northern blot analY5is of c-jun mRNA expression in ovary of rats after treatment for 24 and 48 hours with 2 cm silicone elastomer pellets containing crystalline estradiol-17J3, progesterone, or both. Lanes I to 4, Control, estradiol-17J3, progesterone, and estradiol-17J3 plus progesterone, respectively, exposed for 24 hours; lanes 5 to 8, control, estradiol-17J3, progesterone, and estradiol-17J3 plus progesterone, respectively, exposed for 48 hours.
gene encodes tor a transcription factor 21 ; therefore the inhibition of c-jun by an overdose of estradiol might have affected the transcription of many other unlinked genes. Further investigations should emphasize this issue. By 48 hours of treatment the body appears to eliminate excess estradiol. but the estradiol-1713 level is still above the control level (Table I). This reduction appeared to have enhanced the expression of c1un. This effect of estradiol on c1un expression is observed
in steroidogenic tissues (ovary and adrenal) but not in steroid-responsive tissue (e.g., uterus). It appears that the response of the uterus to tonically administered steroids was different from that of the ovary and adrenal. Estradiol-enhanced induction of c-jun has been previously reported. 22 Our study not only is consistent with these observations but, in addition, shows that the influence of estradiol on c-jun expression is dose dependent and variable among the tissues.
Volume 170, Number 5, Part 1 Am J Obstet Gynecol
REFERENCES 1. Moghissi KS, Syner FN, Evans TN. A composite picture of the menstrual cycle. AM] OBSTET GVNECOL 1972; 114:40513. 2. Goebelsmann U. The menstrual cycle. In: Mischell DR, Cavajan V, eds. Infertility, contraception and reproductive endocrinology. New]ersey, Medical Economics, 1976:6986. 3. Rories C, Spelsberg TC. Ovarian steroid action on gene expression: mechanisms and models. Annu Rev Physiol 1989;51 :653-81. 4. Spelsberg TC, Hora F, Horton M, Ruppert D, Nathans S. Specific DNA binding proteins and DNA sequences involved in steroid honnone regulation of gene expression. In: Thompson F, Papaconstantinou, eds. DNA protein interactions and gene regulation. Austin, Texas: University of Texas Press. 1988. 5. Gorski], Gannon F. Current models of steroid hormone action: a crituque. Annu Rev Biochem 1976;28:425-50. 6. Katzenellenbogan BS. Dynamics of steroid honnone receptor action. Annu Rev Physiol 1980;42: 17 -25. 7. Ransone L]. Verma 1M. Nuclear proto-oncogenes/os and jun. Annu Rev Cell BioI 1990;6:539-57. 8. Weisz A, Bresciani F. Estrogen induces expression of c10s and c-myc protooncogenes in rat uterus. Mol Endocrinol 1988;2:816-24. 9. Webb DK, Moulton BC, Khan SA. Estrogen induced expression of the c-jun protooncogene in the immature and mature rat uterus. Biochem Biophys Res Commun 1990; 168:721-6. 10. Chiappetta C, Kirkland ]L, Loose-Mitchell DS, Murthy L, Stancel GM. Estrogen regulates expression of the Jun family of protooncogenes in the uterus.] Steroid Biochem Mol BioI 1992;69: 121-8. 11. Lau CK, Subramaniam K, Rasmussen K, Spelsberg TC. Rapid inhibition of the c-jun protooncogene expression in avian oviduct by estrogen. Endocrinology 1990;127: 2595-7.
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12. Marrs RP, Vargras ]M, March CM. Correlation of ultrasonic and endocrinologic measurements in human menopausal gonadotropin therapy. AM] OB5TET GVNECOL 1983; 145:417-25. 13. Yen SSC, Martin PL, Burnier AM, Czekala NM, Greaney MO Jr, Callantine MR. Circulating estradiol, estrone and gonadotropin levels following the administration of orally active 17(3-estradiol in postmenopausal women. ] Clin Endocrinol Metab 1975;40:518-21. 14. Nagamani M, Lin T], McDonough PG, Watatani H, McPherson]C, Mahesh VB. Clinical and endocrine studies in menopausal women after estradiol pellet implantation. Obstet Gynecol 1977;50:541-7. 15. Croxatto HB, Diaz S, Pavez M, Miranda P, Brandeis A. Plasma progesterone levels during long-term treatment with medroxyprogesterone acetate Silastic implants. Acta EndocrinoI1982;101:307-11. 16. Hembree WC, Bardin CW, Lipsett MB. A study of estrogen metabolic clearance rates and transfer factors. ] Clin Invest 1969;48:1809-19. 17. Longcope C, Tait ]F. Validity of metabolic clearance and interconversion rates of estrone and 17 -estradiol in normal adults.] Clin Endocrinol Metab 1971;32:481-90. 18. Hembree WC, Bardin CW, Lipsett MB. A study of estrogen metabolic clearance rates and transfer factors. j Clin Invest 1969;48:1809-919. 19. Longcope C, Williams KI. The metabolism of estrogens in normal women after pulse injections of 'H-estradiol and 'H-estrone.] Clin Endocrinol Metab 1974;38:602-7. 20. Little B, Tait jF, Erlenmeyer F. The metabolic clearance rate of progesterone in males and ovariectomized females. j Clin Invest 1966;45:901-12. 21. Macara IG. Oncogenes and cellular signal transduction. Physiol Rev 1989;69:797-820. 22. Chiappetta C, Kirkland ]L, Stancel GM. Estrogen regulates expression of the jun family of protooncogenes in the uterus. ] Steroid Biochem Mol BioI 1992;69: 121-8.
24. 25. 26. 27. 28.
May 1994
Am J Obstet Gynecol
racy and confirmation by gas chromatography/mass spectrometry. j Forensic Sci 1989;34:32-45. Mittleman RE, Cofino jC, Hearn WL. Tissue distribution of cocaine in a pregnant woman. j Forensic Sci 1989;34: 481-6. Fox H, Faulk W. The placenta as an experimental animal. Clin Endocrinol Metab 1981;10:57-72. Fox H. Placenta as a model for organ aging. In: Beaconsfield P, Villee G, eds. Placenta-a neglected experimental animal. Oxford: Pergamon Press, 1979:351-85. Hytten F, Chamberlain G. Clinical physiology in obstetrics. Oxford: Blackwell Scientific Publishers, 1980. Ng W, Miller RK. Transport of nutrients in the early
human placenta: amino acid, creatinine, vitamin B 12 • Trophoblast Res 1983;1:121-34. 29. Dancis j, Levitz M, Katz D, et al. Transfer and metabolism of retinol by the perfused human placenta. Pediatr Res 1992;32:195-9. 30. Fortunato Sj, Bawdon RE, Swan KF, Bryant EC, Sobhi S. Transfer of Timentin (ticarcillin and clavulanic acid) across the in vitro perfused human placenta: comparison with other agents. AM j OBSTET GYNECOL 1992;167:159599. 31. Ahmed MS, Zhou DE, Maulik D, Eldefrawi ME. Characterization of a cocaine binding protein in human placenta. Life Sci 1990;46:553-61.
Differential c-jun gene expression with tonically administered steroids in rat ovary and uterus Martha E. Shelley, MD, Amjad Hossain, PhD, Paul G. McDonough, MD, and Iqbal Khan, PhD
Augusta, Georgia OBJECTIVE: The purpose of this study was to evaluate the induction of the early regulatory gene c-jun in response to tonic exposure to estradiol and progesterone in rat ovary, uterus, and adrenal tissues. STUDY DESIGN: Pellets containing estradiol-17j3, progesterone, and estradiol-17j3 plus progesterone were placed subcutaneously in immature female Sprague-Dawley rats (N = 24). The ovary, uterus, and the adrenal were evaluated for c-jun expression by Northern analysis at 24 and 48 hours. RESULTS: The c-jun messenger ribonucleic acid expression in the ovary and adrenal gland was inhibited with high, non physiologic doses of estradiol in progesterone and was induced with physiologic levels of estradiol. Physiologic levels of progesterone do not appear to influence the expression of c-jun in the ovary or adrenal gland. Uterine c-jun expression to estradiol and progesterone is generally the opposite of that observed in the ovary. CONCLUSION: These findings suggest that there is both tissue and dose specificity of c-jun gene expression in steroidogenic and steroid-responsive tissues when steroid hormones are tonically administered. (AM J OSSTET GVNECOL 1994;170:1410-5.)
Key words: Protooncogene, cjun, estradiol, progesterone The interplay of the two steroid hormones estrogen and progesterone and the growth and differentiation of different tissues in the reproductive system have been the topic of investigation over a long period of time. The physiologic role of steroids in the reproductive process is now fairly well described. I, 2 However, the molecular mechanisms by which steroid hormones and From the Department oj Obstetrics and Gynecology, Medical College of Georgza. Recezved for publicatzon June 8, 1993; reVised August 11, 1993; accepted November 12, 1993. Reprint requests: Iqbal Khan, PhD, Reproductive Endocrinology Sectzon, Cj-134, Department of ObstetriCs and Gynecology, Medlcal College of Georgza, Augusta, GA 30912. Copyright © 1994 by Mosby-Year Book, Inc.
0002-9378/94 $3.00 + 0
1410
6/1/52863
receptors interact to regulate genes participating in the growth or differentiation of reproductive tissues is currently under scrutiny.'·6 Recently the nuclear protooncogenes jun and fos, both components of the AP-l transcription factor, have been demonstrated to play a role in cell proliferation and differentiation and are good candidates for mediators of steroid-induced growth and differentiation. 7 '9 Some studies implicate c-jun as an "early response gene" in the cascade of steroid action on the target tissue. 10, II To date, the response of these protooncogenes to steroid hormones has been evaluated only at the physiologic level of these hormones and at short exposure times. However, in some situations the biologic system may be loaded with steroids for protracted periods far in excess of physio-
Shelley et al.
Volume 170, Number 5, Part 1 Am J Obstet Gynecol
logic levels. For example, ovulation induction in assisted reproductive techniques and menopausal hormonal replacement therapy may expose the body to steroid levels much higher than that produced endogenously.12. 13 Although the physiologic consequences of sudden administration of steroids in the body have been under investigation, the molecular consequences of such steroidal influence is still under scrutiny. In this study we attempted to evaluate the response of one of the early steroid response genes, c-jun, to tonically administered steroids (estradiol, progesterone, or both) in the rat model. Our results show that externally introduced steroids, particularly estradiol, up-regulate steroid metabolism and that the response of protooncogenes c-jun correlates with the (I) type of steroid hormone, (2) circulating level of steroid hormone, and (3) type of tissue. Material and methods
Animals and treatment. Twenty-four immature (29 days old) Sprague-Dawley rats were divided into 24and 48-hour treatment groups. These groups were further subdivided into control, estradiol-l 713 only, progesterone only, and estradiol-1713 plus progesterone subgroups, each containing three animals. With the animals under ether anesthesia 2 cm silicone elastomer pellets containing crystalline estradiol-l 713, progesterone, or both estradiol-1713 and progesterone were placed subcutaneously. The control animals underwent the surgical procedure, but no pellets were placed. At 24 and 48 hours after placement of the pellets the animals were killed, and the ovaries. uterus, and adrenal glands were removed. The tissue was placed immediately into liquid nitrogen and stored at - 80° C until ribonucleic acid (RNA) extraction was performed. RNA isolation and analysis. Total RNA was isolated from the ovaries, uterus, and adrenal glands by the RNAzol method (CinnaiBiotecx). The RNA was quantitated and monitored for purity by evaluating the 260: 280 nm absorption. For the analysis 30 /-Lg of total RNA was fractionated by electrophoresis through a 1.0% agarose-2.2 mol!L formaldehyde gel and transferred to nylon membranes. The nylon membranes were then prehybridized for 1 hour at 65° C in hybridization buffer containing complementary deoxyribonucleic acid probes labeled with phosphorus 32-deoxycytidine triphosphate (3000 Cilmmol) with an oligolabeling kit (Pharmacia, Uppsala, Sweden). The probes used for hybridization were murine c-jun complementary deoxyribonucleic acid and chick-actin complementary deoxyribonucleic acid (used for an internal standard) from American Tissue Culture Collection. After hybridization the membranes were washed under high-stringency conditions and exposed to Kodak X-AR film (Eastman Kodak, Rochester. N.Y.) for 5 days at - 80° C.
1411
The blots were then analyzed by a Shimadzu CS9000 densitometer, and the changes in experimental c-.Jun messenger RNA (mRNA) levels were compared with those in control animals. Hormonal assays. Before death, venous blood was drawn from each animal and centrifuged at 1500 revolutions/min for 10 minutes to separate serum. The serum was stored at - 20° C. The progesterone and rabbit antiprogesterone antibody were provided courtesy of Dr. T. Mills, Augusta, Georgia. The interassay and intraassay variation was 0.5 and 1.0 ng/m!, respectively. The estradiol level in the serum was measured with a standard estradiol assay kit from Diagnostic Products (Los Angeles). The interassay and intraassay variation kit was 1.5 and 5.0 pg/ml, respectively. To confirm a constant release of steroid hormone from the silicone elastomer pellets, the in vitro release pattern of the silicone elastomer pellets was obtained by placing the pellets in 2 ml of phosphate-buffered saline solution for 24, 48, and 72 hours. The progesterone and estradiol levels in the phosphate-buffered saline solution were then measured. Results
Circulating level of steroids. The circulating levels of estradiol and progesterone in rats treated with synthetic steroids subcutaneously administered in 2 cm silicone elastomer pellets is shown in Table I. Serum estradiol levels in estradiol-only treated rats and rats treated with both estradiol and progesterone show an identical pattern. In both groups a marked difference in estradiol level is seen between the 24-hour and 48-hour time periods. By 24 hours the serum estradiol level was elevated fifteenfold compared with the control level but returned toward the control level by 48 hours. The marked difference in estradiol levels between the 24- and 48-hour time periods suggests an up-regulation of estradiol metabolism, because the in vitro release pattern of the silicone elastomer pellets was linear over 72 hours (Fig. 1). In contrast, the serum progesterone level did not reflect any marked difference between the treatment groups (estradiol-l 713 vs progesterone vs estradiol-1713 plus progesterone) over the two time periods (24 vs 48 hours). c-jun mRNA expression. The response of protoonco gene c-jun gene expression in ovary. adrenal gland, and uterus to the synthetic steroid treatment is shown in Figs. 2, 3, and 4, respectively. As seen in Figs. 2 and 5, all three treatment groups of the ovary exhibit a common pattern of expression of c-jun between the 24-hour and 48-hour time periods. The c-jun expression appears to be inhibited in the first 24 hours of exposure to exogenous progesterone and combined estradiol-1713 and progesterone treatment. This inhibition of c-jun occurs with the high level of circulating
1412
May 1994 Am J Obstet Gynecol
Shelley et al.
75 --0--
S
progesterone
- - estradiol
50
-----
o~~~-----+----------+---------~
o
24
48
72
Hours Fig. 1. Measurement of concentration of steroid hormone released from 2 em silicone elastomer pellet after placement in 2 ml of phosphate-buffered saline solution for 24, 48, and 72 hours. Estradiol (trzangles) is expressed as picograms per milliliter, and progesterone (square~) is expressed as nanograms per milliliter at 24, 48, and 72 hours after placement of 2 em silicone elastomer pellet in 2 ml of phosphate-buffered saline solution.
Table I. Circulating levels of estradiol and progesterone in rats treated for 24 and 48 hours with 2 cm silicone elastomer pellets containing crystalline estradiol-1713, progesterone, or both &tradlOl (pg/ml, mean ± SEM) Treatment
Control Estradiol Progesterone Estradiol plus progesterone
24 hr
186 4214 38 3599
± ± ± ±
1.5 4.3 4.1 3.1
estradiol evident at 24 hours (Table I, Fig. 2) in each treatment group. On the other hand, induction of c-jun expression in the 48-hour treatment period is seen when the circulating serum level of steroids is closer to physiologic levels (Table I, Fig. 2). By 48 hours c-jun gene expression was increased 300% in both estradiol1713 and estradiol-1713 plus progesterone treatment groups (Fig. 2). There was no increase or inhibition observed in the ovary in the presence of progesterone, suggesting no synergistic effect of progesterone (Fig. 2). Interestingly, a similar pattern of response to that seen in the ovary was evident in the adrenal, another steroidogenic tissue (Figs. 2 and 3). However, in a steroidresponsive tissue (e.g., uterus) the cjun gene appears to behave differently compared with the response in the ovary and adrenal gland (Figs. 2, 3, and 4). In the uterus there is no sign of c-jun induction; instead suppression is evident in the 48-hour treatment period for all three treatment groups (Fig. 4).
Comment Our study indicates that protracted exposure to steroid hormones results in a differential response of cjun
I
48 hr
182 298 116 308
± 1.7 ± 1.6 ± 4.2 ± 13
Progesterone (ng/ml, mean ± SEM) 24 hr
3.1 2.4 5.4 4.1
± ± ± ±
1.5 3.2 16.4 7.3
I
48 hr
3.4 6.6 3.7 4.6
± ± ± ±
5.3 1.3 6.8 13
expression in steroid-responsive and steroidogenic tissues. This is the first such report of which we are aware that evaluates cjun expression after tonic administration of steroid hormones. The steroid delivery system that uses silicone elastomer pellets for continuous release is well documented in the literature. 14. 15 The pattern of circulating hormone levels observed in our study is consistent with those reported by other investigators. In our study a marked difference in estradiol levels was seen between 24- and 48-hour treatment periods when circulating levels of steroids were analyzed (Table I). Possible up-regulation of steroid metabolism might be responsible for this wide difference in estradiol concentrations between these two time points. Analysis of either the metabolites of estradiol or its clearance from the system will be required to substantiate this speculation. The metabolic clearance of estradiol involves hepatic conjugation, conversion to estrone or estriol, and renal excretion. In previously reported studies factors that influence the rate of clearance of estradiol include dosage, hepatic blood flow, and time of infusion. 16. 17 With dosages of estradiol far in excess of physiologic levels, such as those seen during the first 24 hours of this study, the metabolic
Shelley et al.
Volume 170, Number 5, Part 1 Am J Obstet Gynecol
1413
300 •
250
-- 200
"0
24hrs
048 hrs
'~
0
u
......0 -<
z
~
E
~
150 100 50 0 Estradiol
Estradiol
Progesterone
+ Progesterone
Fig. 2. Graphic representation of cjun mRNA expression in ovary of rats after treatment for 24 and 48 hours with 2 cm silicone elastomer pellets containing crystalline estradiol-17[3, progesterone, or both. Results shown are percent mRNA level of expression of control levels.
200
--
"0 '-
•
24hrs
o 48hrs
150
~
0
u
...... 0
z-<
100
~
E
~
50 0
Estradiol
Progesterone
Estradiol
+ Progesterone
Fig. 3. Graphic representation of c-jun mRNA expression in adrenal gland of rats after treatment for 24 and 48 hours with 2 cm silicone elastomer pellets containing crystalline estradlOl-17[3, progesterone. or both. Results shown are percent mRNA level of expression of control levels.
clearance rate of estradiol has been reported to be significantly increased. IR Also, the clearance rate of estradiol does not appear to reach a steady state for at least 12 hours, in contrast to the 2 hours reported for progesterone. 19,20 The circulating values obtained in our study are consistent with the observations. On the basis of these findings, it can be speculated that a similar pattern of up-regulation may be evident in the case of hormone replacement therapy in the human. However, it is important to note that this study is based on immature rats; thus it remains open to assess whether a similar case is seen in animals that are of varying ages, particularly old age. The important observation of our experiment is that there were differential responses of the cjun gene to
tonically administered steroids. First of all, there was a differential response of the cjun gene to estradiol and progesterone treatment. The influence of progesterone on cjun is insignificant compared with that of estradiol. Poor sensitivity of cjun to progesterone treatment may in part explain this difference. In contrast, the response of cjun to treatment with estradiol was found to be dose dependent and variable among the tissues. The unusually high estradiol level (pharmacologic dose) at the 24-hour treatment period correlates with suppression of c-.Jun expression in all three tissue samples. It appears that when the system was saturated with high levels of estradiol not only complete desensitization of the tissues occurs, but the cellular physiology was also negatively affected. As mentioned earlier, the C-.JUIl
1414 Shelley et al.
May 1994 Am J Ob,tet Gynecol
150 .
as....
....>
c 0
u
24 hrs
048 hrs
100
a
<:
:z 0:::
E
50
~
0
Estradiol
Estradiol +
Progesterone
Progesterone Fig. 4. Graphic representation of c-jun mRNA expression in uterus of rats after treatment for 24 and 48 hours with 2 cm silicone elastomer pellets containing crystalline estradlOl-17J3, progesterone, or both. Results ~hown are percent mRNA level of expression of control levels.
2
3
4
5
6
7
8
c-jun
2.6 kb
actin
2.0 kb
24 hours
48 hours
Fig. 5. Northern blot analY5is of c-jun mRNA expression in ovary of rats after treatment for 24 and 48 hours with 2 cm silicone elastomer pellets containing crystalline estradiol-17J3, progesterone, or both. Lanes I to 4, Control, estradiol-17J3, progesterone, and estradiol-17J3 plus progesterone, respectively, exposed for 24 hours; lanes 5 to 8, control, estradiol-17J3, progesterone, and estradiol-17J3 plus progesterone, respectively, exposed for 48 hours.
gene encodes tor a transcription factor 21 ; therefore the inhibition of c-jun by an overdose of estradiol might have affected the transcription of many other unlinked genes. Further investigations should emphasize this issue. By 48 hours of treatment the body appears to eliminate excess estradiol. but the estradiol-1713 level is still above the control level (Table I). This reduction appeared to have enhanced the expression of c1un. This effect of estradiol on c1un expression is observed
in steroidogenic tissues (ovary and adrenal) but not in steroid-responsive tissue (e.g., uterus). It appears that the response of the uterus to tonically administered steroids was different from that of the ovary and adrenal. Estradiol-enhanced induction of c-jun has been previously reported. 22 Our study not only is consistent with these observations but, in addition, shows that the influence of estradiol on c-jun expression is dose dependent and variable among the tissues.
Volume 170, Number 5, Part 1 Am J Obstet Gynecol
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