Volume 167 Number 6
22. Aubert ]F, Burnier M, Waeber B, Nussberger ], Brunner HR. Nicotine-induced release of vasopressin in the conscious rat: role of opioid pep tides and hemodynamic effects.] Phannacol Exp Ther 1987;243:681-5. 23. Irion GL, Mack CE, Clark KE. Fetal hemodynamic and fetoplacental vascular response to exogenous arginine vasopressin. AM] OBSTET GYNECOL 1990;162:1115-20. 24. Waeber B, Schaller M-D, Nussberger ], Bussien ]-P, Hofbauer KG, Brunner HR. Skin blood flow reduction induced by cigarette smoking: role of vasopressin. Am ] Physiol 1984;247:H895-901.
Nicotine and umbilical blood flow
25. Busacca M, Balconi G, Pietra A, Vergara-Dauden M, de Gaetano G, Dejana E. Maternal smoking and prostacyclin production by cultured endothelial cells from umbilical arteries. AM] OBSTET GYNECOL 1984;148:1127-30. 26. Lindblad A, Marsal K, Andersson K-E. Effect of nicotine on human fetal blood flow. Obstet Gynecol 1988;72:37182. 27. Resnick R, Conover WB, Key TC, Van Vunakis H. Uterine blood flow and catecholamine response to repetitive nicotine exposure in the pregnant ewe. AM] OSSTET GYNECOL 1985; 151 :885-91.
The concentration of estrogen receptors in rabbit uterine myocytes decreases in culture Yoel Sadovsky, MD, R. Kirk Riemer, PhD, and James M. Roberts, MD
San Francisco, California OBJECTIVE: The purpose of this study was to determine whether the concentration of estrogen receptors in cultured myocytes is preserved after dispersion. STUDY DESIGN: Primary myocytes were prepared from rabbit myometrium by collagenase dispersion after removing the endometrium and were isolated with Percoll density gradients. The cells were assayed for estrogen receptor concentration at intervals after dispersion by means of a whole-cell binding assay. Unpaired t test was used for comparisons. RESULTS: The concentration of estrogen receptors on the first day after dispersion was 12,058 ± 1096 sites per cell (mean ± SEM) and decreased to 4389 ± 1223 site per cell within 9 to 14 days after dispersion (63% decline, p < 0.001). A similar decrease was observed when 2 nmol/L estradiol was present in the medium. CONCLUSION: The concentration of estrogen receptors in isolated rabbit uterine myocytes decreases after dispersion. This may partly explain the difficulty of demonstrating in vitro estrogen effects on myocytes, which are well established in vivo. (AM J OBSTET GYNECOL 1992;167:1631-5.)
Key words: Rabbit uterine myocytes, estrogen, estrogen receptors Estrogen has an important role in regulation of uterine growth and differentiation. Endogenous estrogen levels or estrogen administration to animals of several species enhance mitogenesis i • 2 and regulate the concentration of uterine proteins. Examples include
From the Department of Obstetrics, Gynecology, and Reproductive Sciences, and the Cardiovascular Research Institute, University of California, San Francisco. Supported by an American College of Obstetricians and Gynecologists-Ortho Pharmaceuticals grant (Y.S.), and United States Public Health Service grants HD26152 (R.K.R.) and HD21785 (J.M.R.). Presented at the Thirty-ninth Annual Meeting of the Society for Gynecologic Investigation, San Antonio, Texas, March 18-21,1992. Reprint requests: Yoel Sadovsky, MD, Division of Maternal-Fetal Medicine, Box 0550, HSE 1462, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, San Francisco, CA 94143-0550. 6/6/41928
progesterone receptors,3. 4 enzymes such as creatine kinase B,5 oncogenes,6 and growth factors such as epidermal growth factor and its receptors. 7 . 8 Estrogen also regulates myometrial contractile functions, demonstrated by the positive effect of estrogen on a,- and a 2 -adrenergic pathways,g· 10 the negative effect on ~-adrenergic pathways, II and the increase in oxytocin receptors and gap junction concentration in response to estradiol administration. 12. 13 Understanding the cellular mechanisms underlying estrogen effects would be facilitated by an estrogen responsive in vitro system, which could test whether specific estrogen effects are direct or dependent on endocrine, paracrine, or autocrine mediators. However, estrogen effects on primary myocytes in vitro have been difficult to demonstrate. Because estrogen responsiveness of isolated myocytes depends on the maintenance 1631
1632 Sadovsky, Riemer, and Roberts
of estrogen receptor concentration in these cells, we determined whether estrogen receptor concentration in isolated cultured primary myocytes is preserved after dispersion, and we report a decrease in these receptors during culture. Material and methods
Preparation of myocytes. Six mature, nonpregnant New Zealand White female rabbits were used for the study. The animals were treated for 4 days with daily injections of 50 mg/kg estradiol benzoate. On the fifth day the animals were killed by overdose of pentobarbital sodium (100 mgikg given intravenously), and the uteri were removed under sterile conditions and placed briefly in ice-cold Hanks' balanced salt solution. Each uterine hom was stripped of fat and mesentery, and was opened longitudinally; the endometrium was removed by scraping. The myometrial tissue was vigorously washed and minced to pieces of 1 to 2 mm, and the minces were resuspended in 20 ml of Dulbecco's modified Eagle's medium with 4.5 gm/L glucose, 50 mmoVL (N -[2 -hydroxyethy l]piperazine-N I -[2-ethanesulfonic acid]) (pH 7.4), 0.05% bovine serum albumin 5 mg/ml insulin, transferrin, 5 ng/ml selenium (Sigma, St. Louis), 1% penicillin and streptomycin, and 500 ng/ml Fungizone. The medium also contained collagenase II (1 mg/ml) and IV (1 mg/ml) (Boehringer Mannheim, Indianapolis) and 0.1 mg/ml deoxyribonuclease (Boehringer Mannheim, Mannheim, Germany). The minces were incubated in this medium at 37° C with continuous stirring. Intact minces remaining after 4 hours' incubation were subjected to repeat digestion for 4 additional hours. The dispersed cells were collected and pooled in Hanks' balanced salt solution, washed, then resuspended in 8 ml of Hanks' balanced salt solution containing 0.5 mg/ml bovine serum albumin and 100 mg/ml deoxyribonuclease. Aliquots of 1 ml were loaded onto 8 ml gradients of 35% Percoll (Sigma) diluted with Hanks' balanced salt solution that contained the same concentration of bovine serum albumin and deoxyribonuclease. The gradients were preformed by centrifugation at 30,000g for 15 minutes. Myocytes were separated from red blood cells and debris by centrifugation at 2000g and washed, and then the number of viable cells was determined by trypan blue dye exclusion. Viability was > 80%. The harvested cells were plated in 24-well plates (Falcon, Becton Dickinson and Co., Lincoln Park, N.].) in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 100 ng/ml insulin, 1% penicillin and streptomycin, and 500 ng/ml Fungizone and incubated at 37° C in a humidified atmosphere of 95% air and 5% carbon dioxide with medium changes every 3 days. To suppress fibroblast proliferation, o-valine (9.4 mg/ml) was substituted for L-valine in the culture me-
December 1992 Am J Obstet Gynecol
dium. 14 In some experiments 2 nmoVL estradiol (Sigma) in ethanol was added to the medium, keeping ethanol concentration s; 0.1 %. One day before estrogen receptor assay, the cells were washed and the medium changed to phenol red-free Dulbecco's modified Eagle's medium containing 10% charcoal dextrantreated fetal calf serum. Estrogen receptor-binding assay. All binding assays were performed in 24-well plates. Estrogen receptor concentration was determined in triplicate by means of a whole-cell binding assay, described by Taylor et al. 15 The medium was replaced by phenol red-free Dulbecco's modified Eagle's medium containing 0.1% bovine serum albumin and 1 nmoVL tritiated estradiol (specific activity 92.5 Ci!mmol, Du Pont, Boston) in the presence or absence of 100 nmoVL unlabeled estradiol, to determine nonspecific binding. The concentration of tritiated estradiol is at least five times the measured dissociation constant for the receptor, and thus sufficient to saturate available receptors. After 90 minutes of incubation at 37° C, the medium was changed to phosphate buffered saline solution with 0.5% bovine serum albumin for 30 minutes at room temperature. The cells were washed twice in ice-cold phosphate-buffered saline solution, and the labeled ligand was extracted with 100% ethanol and counted by liquid scintillator (Beckman, Irvine, Calif.). Nonspecific binding was 40% to 60% of total binding, with a higher fraction of nonspecific binding when receptor concentration was low. The validity of determining receptor density increase with a single concentration of radioligand was confirmed by saturation analysis, in which the cells were incubated with increasing concentrations (0.015 to 2.5 nmoVL) of radioligand, and receptor concentration was determined by means of a nonlinear, iterative, curve-fitting program. 16 For cell counts the myocytes were diluted in trypan blue-containing buffer, and viable cells, which excluded the dye, were counted with a hemocytometer. Viability was > 80%. Results were calculated as receptor sites per cell. Data are presented as mean ± SEM. For comparisons unpaired two-tailed t test was used. Significance was determined at p < 0.05. The study was approved by the Committee on Animal Research at The University of California, San Francisco, and the institutional guidelines for the care and use of the animals were followed. Results
We measured estrogen receptor concentration on days 1, 3, 6, 9, and 14 after dispersion. The results for each of the six rabbits, showing a variable rate of estrogen receptor concentration decline during culture, are depicted in Fig. L Mean receptor concentrations after 1 day in culture and at time of maximal decrease is shown in Fig. 2. The mean concentration of estrogen
Volume 167 Number 6
Estrogen receptors in cultured uterine myocytes
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Days after dispersal Fig. 1. Concentration of estrogen receptors (expressed as sites per cell) in cultured rabbit uterine myocytes, measured at intervals after dispersion. Each line represents one animal (n = 6).
---
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Fig. 2. Mean estrogen receptor concentration (expressed as sites per cell) measured in cultured rabbit myocytes on the first day and the lowest concentration measured between 9 and 14 days after dispersion, in the absence (p < 0.001, n = 6) or presence (p < 0.025, n = 3) of estradiol (£2) (2 nmoVL) in the medium. Difference between two lowest measured concentrations was not significant (p = 0.47).
receptors 24 hours after dispersion was 12058 ± 1096 sites per cell and decreased to a mean of 4389 ± 1223 sites per cell (63% decline, p < 0.001). Because of the variable rate of decline, this value reflects the lowest
receptor concentration measured between 9 and 14 days after dispersion. We also verified the concentration of estrogen receptors on days I and 14 after dispersion in representative experiments with saturation isotherms
1634 Sadovsky. Riemer. and Roberts
and found a concentration decrease from 10713 to 3392 sites per cell, in good agreement with the singlepoint assays. Because all rabbits were treated with estradiol before dispersal, we determined whether withdrawal from estrogen was responsible for this receptor concentration decrease. We performed experiments in three rabbits in which estradiol (2 nmoVL) was added to the culture medium and compared this with cells cultured without estradiol. The results are shown in Fig. 2. Estrogen receptor concentration again decreased during 14 days in culture, from 12,423 ± 1459 to 5864 ± 1174 sites per cell (52% decline, p < 0.025). Maximum decrease measured was similar to the decrease in cultures without added estrogen (p = 0.47). Estrogen receptor concentration did not correlate with the degree of cellular confluence, which ranged from 150,000 to 400,000 cells per well in the assay. To verify that the myocytes were not overgrown by fibroblasts, cultured cells were stained for a-smooth muscle actin after 10 days in culture. The vast majority of the cells stained positive, confirming the preponderance of myocytes over other cell types.
Comment Although the effects of estrogen on myometrial growth and differentiated responses have been extensively demonstrated in vivo, similar effects have been difficult to reproduce in isolated myocytes. In this study we propose one mechanism for this difficulty. Our results show that estrogen receptor concentration in isolated rabbit myocytes declines in culture, reaching a third of the initial level within 2 weeks. This decline was not prevented by the presence of 2 nmoVL estradiol in the culture medium, suggesting that the lower receptor concentration cannot be explained by withdrawal from endogenous estrogen. The concentration of estrogen receptors found in the first day after dispersion was consistent with the value reported by others, 17. 18 and this concentration was similar whether the receptor concentration was measured in plated cells or in isolated cells before plating (results not shown). Although Katzenellenbogen and Gorski l9 have found an 82% decrease in the ability of estrogen to induce a specific protein in mixed rat uterine cell cultures by 8 hours, Krosl et al. 20 have found in a similar preparation that estrogen receptor concentration declined by 75% within only 2 to 3 weeks. Sumida and Pasqualini,21 studying fetal guinea pig myometrial-stromal cells, reported that estrogen responsiveness declined by 80% within 16 days in culture. In addition to species differences, the variability in the ability to maintain functional receptors may depend on medium components, frequency of medium changes, and interval between dispersion and measurement, as demonstrated by Kassis et al. 17
December 1992 Am J Obstet Gynecol
In our experiments we used isolated myocytes, because other uterine cell types may complicate the system by virtue of their own estrogen receptors. 18. 22 Moreover, uterine epithelium appears to enhance myometrial smooth muscle differentiation. 23 Therefore interaction of endometrial cells with myocytes may contribute to the maintenance of estrogen responsiveness. Nevertheless, the fact that others have found similar results with mixed cells makes it unlikely that removal of myocytes from paracrine interactions with epithelial cells explains the decrease in receptor concentration. In summary, the concentration of estrogen receptors in cultured rabbit myocytes decreases after dispersion, and this decrease was not prevented by added estradiol. Although the mechanism of receptor concentration decline is still unclear, it may partly explain the difficulty in demonstrating estrogen effects in cultured primary myocytes in vitro. It should be noted, however, that several estradiol-induced responses we observed in vivo, such as an increase in oxytocin receptors 24 and adrenergic-mediated production of inositol phosphates,IO were not demonstrated in vitro even within 1 to 2 days after dispersion, before the expected decline in estrogen receptor concentration. This suggests therefore that paracrine interaction by endometrial or stromal cells may also be important for maintenance of estrogen responsiveness in cultured myocytes. We thank Dr. Alan Goldfien for careful reading and thoughtful suggestions in the preparation of this manuscript. REFERENCES 1. Lee AE. Cell division and DNA synthesis in the mouse uterus during continuous estrogen treatment. J Endocrinol 1972;55:507-13. 2. Chen L, Lindner HR, Lancet M. Mitogenic action of oestradiol-17~ on human myometrial and endometrial cells in long-term tissue cultures. J Endocrinol 1973;59: 87-97. 3. Aronica SM, Katzenellenbogen BS. Progesterone receptor regulation in uterine cells: stimulation by estrogen, cyclic adenosine 3',5' -monophosphate, and insulin-like growth factor I and suppression by antiestrogens and protein kinase inhibitors. Endocrinology 1991;128:2045-52. 4. Dix Cj, Jordan VC. Modulation of rat uterine steroid hormone receptors by estrogen and antiestrogen. Endocrinology 1980; 107 :20 11-20. 5. Pentecost BT, Mattheiss L, Dickerman HW, Kumar A. Estrogen regulation of creatine kinase-B in the rat uterus. Mol Endocrinol 1990;4: 1000-1 O. 6. Murphy LJ, Murphy Le, Friesen HG. Estrogen induction of N-myc and c-myc proto-oncogene expression in the rat uterus. Endocrinology 1987;120:1882-8. 7. Heut-Hudson YM, Chakraborty C, De SK, Suzuki Y, Andrews GK, Dey SK. Estrogen regulates synthesis of epidermal growth factor in mouse uterine epithelial cells. Mol Endocrinol 1990;4:510-23. 8. Mukku VR, Stancel GM. Regulation of epidermal growth factor by estrogen. J Bioi Chern 1985;260:9820-4. 9. Roberts JM, Riemer RK, Bottari SP, Wu YY, Goldfien A. Hormonal regulation myometrial adrenergic responses: the receptor and beyond. J Dev Physiol 1989;11:125-34. 10. Riemer RK, Goldfien A, Roberts JM. Estrogen increases
Volume 167 Number 6
11.
12. 13. 14. 15.
16. 17.
Estrogen receptors in cultured uterine myocytes
adrenergic but not cholinergic-mediated production of inositol phosphates in rabbit uterus. Mol Pharmacol1987; 32:663-8. Riemer RK, Wu YV, Bottari SP, Jacobs MM, Goldfien A, Roberts ]M. Estrogen reduces beta-adrenoceptor-mediated cAMP production and the concentration of the guanyl nucleotide-regulatory protein, G" in rabbit myometrium. Mol Pharmacol 1988;33:389-95. Nissenson R, Flouret G, Hechter O. Opposing effects of estradiol and progesterone on oxytocin receptors in rabbit uterus. Proc Nat! Acad Sci USA 1978;75:2044-8. MacKenzie LW, Garfield RE. Hormonal control of gap junctions in the myometrium. Am] PhysioI1985;17:C296308. Gilbert SF, Migeon BR. D-Valine as a selective agent for normal human and rodent epithelial cells in culture. Cell 1975;5:11-7. Taylor CM, Blanchard BB, Zava DT. A simple method to determine whole cell uptake of radiolabelled estrogen and progesterone and their subcellular localization in breast cancer cell line in monolayer culture. ] Steroid Biochem 1984;20: 1983-8. Murlas C, Nadel]A, Roberts]M. The muscarinic receptors ofaiIWay smooth muscle.] Appl PhysioI1982;52:1084-91. Kassis ]A, Walent ]H, Gorski J. Estrogen receptors in rat
18. 19. 20. 21. 22.
23. 24.
1635
uterine cell cultures: effects of medium on receptor concentration. Endocrinology 1984; 115:762-9. McCormack SA, Glasser SR. Differential response of individual uterine cell types from immature rats treated with estradiol. Endocrinology 1980; 106: 1634-49. Katzenellenbogen BS, Gorski J. Estrogen action in vitro. ] Bioi Chern 1972;247: 1299-1305. Krosl], Breskvar K, Hudnik-Plevnik T. Prolonged cultivation of rat uterine cells with preserved estrogen responsiveness.] Steroid Biochem 1989;33:189-94. Sumida C, Pasqualini JR. Estrogen responsiveness of fetal guinea pig uterine cells in culture. J Steroid Biochem 1986;24:231-4. Zaino ]R, Clarke CL, Feil PD, Satyaswaroop PG. Differential distribution of estrogen and progesterone receptors in rabbit uterus detected by dual immunofluorescence. Endocrinology 1989; 125:2728-34. Cunha GR, Young P, Brody JR. Role of uterine epithelium in the development of myometrial smooth muscles cells. Bioi Reprod 1989;40:861-71. Jacobson L, Riemer RK, GoldfienAC, Lykins D, Siiteri PK, Roberts JM. Rabbit myometrial oxytocin and a 2 -adrenergic receptors are increased by estrogen but are differentially regulated by progesterone. Endocrinology 1987; 120: 1184-9.
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22. Aubert ]F, Burnier M, Waeber B, Nussberger ], Brunner HR. Nicotine-induced release of vasopressin in the conscious rat: role of opioid pep tides and hemodynamic effects.] Phannacol Exp Ther 1987;243:681-5. 23. Irion GL, Mack CE, Clark KE. Fetal hemodynamic and fetoplacental vascular response to exogenous arginine vasopressin. AM] OBSTET GYNECOL 1990;162:1115-20. 24. Waeber B, Schaller M-D, Nussberger ], Bussien ]-P, Hofbauer KG, Brunner HR. Skin blood flow reduction induced by cigarette smoking: role of vasopressin. Am ] Physiol 1984;247:H895-901.
Nicotine and umbilical blood flow
25. Busacca M, Balconi G, Pietra A, Vergara-Dauden M, de Gaetano G, Dejana E. Maternal smoking and prostacyclin production by cultured endothelial cells from umbilical arteries. AM] OBSTET GYNECOL 1984;148:1127-30. 26. Lindblad A, Marsal K, Andersson K-E. Effect of nicotine on human fetal blood flow. Obstet Gynecol 1988;72:37182. 27. Resnick R, Conover WB, Key TC, Van Vunakis H. Uterine blood flow and catecholamine response to repetitive nicotine exposure in the pregnant ewe. AM] OSSTET GYNECOL 1985; 151 :885-91.
The concentration of estrogen receptors in rabbit uterine myocytes decreases in culture Yoel Sadovsky, MD, R. Kirk Riemer, PhD, and James M. Roberts, MD
San Francisco, California OBJECTIVE: The purpose of this study was to determine whether the concentration of estrogen receptors in cultured myocytes is preserved after dispersion. STUDY DESIGN: Primary myocytes were prepared from rabbit myometrium by collagenase dispersion after removing the endometrium and were isolated with Percoll density gradients. The cells were assayed for estrogen receptor concentration at intervals after dispersion by means of a whole-cell binding assay. Unpaired t test was used for comparisons. RESULTS: The concentration of estrogen receptors on the first day after dispersion was 12,058 ± 1096 sites per cell (mean ± SEM) and decreased to 4389 ± 1223 site per cell within 9 to 14 days after dispersion (63% decline, p < 0.001). A similar decrease was observed when 2 nmol/L estradiol was present in the medium. CONCLUSION: The concentration of estrogen receptors in isolated rabbit uterine myocytes decreases after dispersion. This may partly explain the difficulty of demonstrating in vitro estrogen effects on myocytes, which are well established in vivo. (AM J OBSTET GYNECOL 1992;167:1631-5.)
Key words: Rabbit uterine myocytes, estrogen, estrogen receptors Estrogen has an important role in regulation of uterine growth and differentiation. Endogenous estrogen levels or estrogen administration to animals of several species enhance mitogenesis i • 2 and regulate the concentration of uterine proteins. Examples include
From the Department of Obstetrics, Gynecology, and Reproductive Sciences, and the Cardiovascular Research Institute, University of California, San Francisco. Supported by an American College of Obstetricians and Gynecologists-Ortho Pharmaceuticals grant (Y.S.), and United States Public Health Service grants HD26152 (R.K.R.) and HD21785 (J.M.R.). Presented at the Thirty-ninth Annual Meeting of the Society for Gynecologic Investigation, San Antonio, Texas, March 18-21,1992. Reprint requests: Yoel Sadovsky, MD, Division of Maternal-Fetal Medicine, Box 0550, HSE 1462, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, San Francisco, CA 94143-0550. 6/6/41928
progesterone receptors,3. 4 enzymes such as creatine kinase B,5 oncogenes,6 and growth factors such as epidermal growth factor and its receptors. 7 . 8 Estrogen also regulates myometrial contractile functions, demonstrated by the positive effect of estrogen on a,- and a 2 -adrenergic pathways,g· 10 the negative effect on ~-adrenergic pathways, II and the increase in oxytocin receptors and gap junction concentration in response to estradiol administration. 12. 13 Understanding the cellular mechanisms underlying estrogen effects would be facilitated by an estrogen responsive in vitro system, which could test whether specific estrogen effects are direct or dependent on endocrine, paracrine, or autocrine mediators. However, estrogen effects on primary myocytes in vitro have been difficult to demonstrate. Because estrogen responsiveness of isolated myocytes depends on the maintenance 1631
1632 Sadovsky, Riemer, and Roberts
of estrogen receptor concentration in these cells, we determined whether estrogen receptor concentration in isolated cultured primary myocytes is preserved after dispersion, and we report a decrease in these receptors during culture. Material and methods
Preparation of myocytes. Six mature, nonpregnant New Zealand White female rabbits were used for the study. The animals were treated for 4 days with daily injections of 50 mg/kg estradiol benzoate. On the fifth day the animals were killed by overdose of pentobarbital sodium (100 mgikg given intravenously), and the uteri were removed under sterile conditions and placed briefly in ice-cold Hanks' balanced salt solution. Each uterine hom was stripped of fat and mesentery, and was opened longitudinally; the endometrium was removed by scraping. The myometrial tissue was vigorously washed and minced to pieces of 1 to 2 mm, and the minces were resuspended in 20 ml of Dulbecco's modified Eagle's medium with 4.5 gm/L glucose, 50 mmoVL (N -[2 -hydroxyethy l]piperazine-N I -[2-ethanesulfonic acid]) (pH 7.4), 0.05% bovine serum albumin 5 mg/ml insulin, transferrin, 5 ng/ml selenium (Sigma, St. Louis), 1% penicillin and streptomycin, and 500 ng/ml Fungizone. The medium also contained collagenase II (1 mg/ml) and IV (1 mg/ml) (Boehringer Mannheim, Indianapolis) and 0.1 mg/ml deoxyribonuclease (Boehringer Mannheim, Mannheim, Germany). The minces were incubated in this medium at 37° C with continuous stirring. Intact minces remaining after 4 hours' incubation were subjected to repeat digestion for 4 additional hours. The dispersed cells were collected and pooled in Hanks' balanced salt solution, washed, then resuspended in 8 ml of Hanks' balanced salt solution containing 0.5 mg/ml bovine serum albumin and 100 mg/ml deoxyribonuclease. Aliquots of 1 ml were loaded onto 8 ml gradients of 35% Percoll (Sigma) diluted with Hanks' balanced salt solution that contained the same concentration of bovine serum albumin and deoxyribonuclease. The gradients were preformed by centrifugation at 30,000g for 15 minutes. Myocytes were separated from red blood cells and debris by centrifugation at 2000g and washed, and then the number of viable cells was determined by trypan blue dye exclusion. Viability was > 80%. The harvested cells were plated in 24-well plates (Falcon, Becton Dickinson and Co., Lincoln Park, N.].) in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 100 ng/ml insulin, 1% penicillin and streptomycin, and 500 ng/ml Fungizone and incubated at 37° C in a humidified atmosphere of 95% air and 5% carbon dioxide with medium changes every 3 days. To suppress fibroblast proliferation, o-valine (9.4 mg/ml) was substituted for L-valine in the culture me-
December 1992 Am J Obstet Gynecol
dium. 14 In some experiments 2 nmoVL estradiol (Sigma) in ethanol was added to the medium, keeping ethanol concentration s; 0.1 %. One day before estrogen receptor assay, the cells were washed and the medium changed to phenol red-free Dulbecco's modified Eagle's medium containing 10% charcoal dextrantreated fetal calf serum. Estrogen receptor-binding assay. All binding assays were performed in 24-well plates. Estrogen receptor concentration was determined in triplicate by means of a whole-cell binding assay, described by Taylor et al. 15 The medium was replaced by phenol red-free Dulbecco's modified Eagle's medium containing 0.1% bovine serum albumin and 1 nmoVL tritiated estradiol (specific activity 92.5 Ci!mmol, Du Pont, Boston) in the presence or absence of 100 nmoVL unlabeled estradiol, to determine nonspecific binding. The concentration of tritiated estradiol is at least five times the measured dissociation constant for the receptor, and thus sufficient to saturate available receptors. After 90 minutes of incubation at 37° C, the medium was changed to phosphate buffered saline solution with 0.5% bovine serum albumin for 30 minutes at room temperature. The cells were washed twice in ice-cold phosphate-buffered saline solution, and the labeled ligand was extracted with 100% ethanol and counted by liquid scintillator (Beckman, Irvine, Calif.). Nonspecific binding was 40% to 60% of total binding, with a higher fraction of nonspecific binding when receptor concentration was low. The validity of determining receptor density increase with a single concentration of radioligand was confirmed by saturation analysis, in which the cells were incubated with increasing concentrations (0.015 to 2.5 nmoVL) of radioligand, and receptor concentration was determined by means of a nonlinear, iterative, curve-fitting program. 16 For cell counts the myocytes were diluted in trypan blue-containing buffer, and viable cells, which excluded the dye, were counted with a hemocytometer. Viability was > 80%. Results were calculated as receptor sites per cell. Data are presented as mean ± SEM. For comparisons unpaired two-tailed t test was used. Significance was determined at p < 0.05. The study was approved by the Committee on Animal Research at The University of California, San Francisco, and the institutional guidelines for the care and use of the animals were followed. Results
We measured estrogen receptor concentration on days 1, 3, 6, 9, and 14 after dispersion. The results for each of the six rabbits, showing a variable rate of estrogen receptor concentration decline during culture, are depicted in Fig. L Mean receptor concentrations after 1 day in culture and at time of maximal decrease is shown in Fig. 2. The mean concentration of estrogen
Volume 167 Number 6
Estrogen receptors in cultured uterine myocytes
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Days after dispersal Fig. 1. Concentration of estrogen receptors (expressed as sites per cell) in cultured rabbit uterine myocytes, measured at intervals after dispersion. Each line represents one animal (n = 6).
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Fig. 2. Mean estrogen receptor concentration (expressed as sites per cell) measured in cultured rabbit myocytes on the first day and the lowest concentration measured between 9 and 14 days after dispersion, in the absence (p < 0.001, n = 6) or presence (p < 0.025, n = 3) of estradiol (£2) (2 nmoVL) in the medium. Difference between two lowest measured concentrations was not significant (p = 0.47).
receptors 24 hours after dispersion was 12058 ± 1096 sites per cell and decreased to a mean of 4389 ± 1223 sites per cell (63% decline, p < 0.001). Because of the variable rate of decline, this value reflects the lowest
receptor concentration measured between 9 and 14 days after dispersion. We also verified the concentration of estrogen receptors on days I and 14 after dispersion in representative experiments with saturation isotherms
1634 Sadovsky. Riemer. and Roberts
and found a concentration decrease from 10713 to 3392 sites per cell, in good agreement with the singlepoint assays. Because all rabbits were treated with estradiol before dispersal, we determined whether withdrawal from estrogen was responsible for this receptor concentration decrease. We performed experiments in three rabbits in which estradiol (2 nmoVL) was added to the culture medium and compared this with cells cultured without estradiol. The results are shown in Fig. 2. Estrogen receptor concentration again decreased during 14 days in culture, from 12,423 ± 1459 to 5864 ± 1174 sites per cell (52% decline, p < 0.025). Maximum decrease measured was similar to the decrease in cultures without added estrogen (p = 0.47). Estrogen receptor concentration did not correlate with the degree of cellular confluence, which ranged from 150,000 to 400,000 cells per well in the assay. To verify that the myocytes were not overgrown by fibroblasts, cultured cells were stained for a-smooth muscle actin after 10 days in culture. The vast majority of the cells stained positive, confirming the preponderance of myocytes over other cell types.
Comment Although the effects of estrogen on myometrial growth and differentiated responses have been extensively demonstrated in vivo, similar effects have been difficult to reproduce in isolated myocytes. In this study we propose one mechanism for this difficulty. Our results show that estrogen receptor concentration in isolated rabbit myocytes declines in culture, reaching a third of the initial level within 2 weeks. This decline was not prevented by the presence of 2 nmoVL estradiol in the culture medium, suggesting that the lower receptor concentration cannot be explained by withdrawal from endogenous estrogen. The concentration of estrogen receptors found in the first day after dispersion was consistent with the value reported by others, 17. 18 and this concentration was similar whether the receptor concentration was measured in plated cells or in isolated cells before plating (results not shown). Although Katzenellenbogen and Gorski l9 have found an 82% decrease in the ability of estrogen to induce a specific protein in mixed rat uterine cell cultures by 8 hours, Krosl et al. 20 have found in a similar preparation that estrogen receptor concentration declined by 75% within only 2 to 3 weeks. Sumida and Pasqualini,21 studying fetal guinea pig myometrial-stromal cells, reported that estrogen responsiveness declined by 80% within 16 days in culture. In addition to species differences, the variability in the ability to maintain functional receptors may depend on medium components, frequency of medium changes, and interval between dispersion and measurement, as demonstrated by Kassis et al. 17
December 1992 Am J Obstet Gynecol
In our experiments we used isolated myocytes, because other uterine cell types may complicate the system by virtue of their own estrogen receptors. 18. 22 Moreover, uterine epithelium appears to enhance myometrial smooth muscle differentiation. 23 Therefore interaction of endometrial cells with myocytes may contribute to the maintenance of estrogen responsiveness. Nevertheless, the fact that others have found similar results with mixed cells makes it unlikely that removal of myocytes from paracrine interactions with epithelial cells explains the decrease in receptor concentration. In summary, the concentration of estrogen receptors in cultured rabbit myocytes decreases after dispersion, and this decrease was not prevented by added estradiol. Although the mechanism of receptor concentration decline is still unclear, it may partly explain the difficulty in demonstrating estrogen effects in cultured primary myocytes in vitro. It should be noted, however, that several estradiol-induced responses we observed in vivo, such as an increase in oxytocin receptors 24 and adrenergic-mediated production of inositol phosphates,IO were not demonstrated in vitro even within 1 to 2 days after dispersion, before the expected decline in estrogen receptor concentration. This suggests therefore that paracrine interaction by endometrial or stromal cells may also be important for maintenance of estrogen responsiveness in cultured myocytes. We thank Dr. Alan Goldfien for careful reading and thoughtful suggestions in the preparation of this manuscript. REFERENCES 1. Lee AE. Cell division and DNA synthesis in the mouse uterus during continuous estrogen treatment. J Endocrinol 1972;55:507-13. 2. Chen L, Lindner HR, Lancet M. Mitogenic action of oestradiol-17~ on human myometrial and endometrial cells in long-term tissue cultures. J Endocrinol 1973;59: 87-97. 3. Aronica SM, Katzenellenbogen BS. Progesterone receptor regulation in uterine cells: stimulation by estrogen, cyclic adenosine 3',5' -monophosphate, and insulin-like growth factor I and suppression by antiestrogens and protein kinase inhibitors. Endocrinology 1991;128:2045-52. 4. Dix Cj, Jordan VC. Modulation of rat uterine steroid hormone receptors by estrogen and antiestrogen. Endocrinology 1980; 107 :20 11-20. 5. Pentecost BT, Mattheiss L, Dickerman HW, Kumar A. Estrogen regulation of creatine kinase-B in the rat uterus. Mol Endocrinol 1990;4: 1000-1 O. 6. Murphy LJ, Murphy Le, Friesen HG. Estrogen induction of N-myc and c-myc proto-oncogene expression in the rat uterus. Endocrinology 1987;120:1882-8. 7. Heut-Hudson YM, Chakraborty C, De SK, Suzuki Y, Andrews GK, Dey SK. Estrogen regulates synthesis of epidermal growth factor in mouse uterine epithelial cells. Mol Endocrinol 1990;4:510-23. 8. Mukku VR, Stancel GM. Regulation of epidermal growth factor by estrogen. J Bioi Chern 1985;260:9820-4. 9. Roberts JM, Riemer RK, Bottari SP, Wu YY, Goldfien A. Hormonal regulation myometrial adrenergic responses: the receptor and beyond. J Dev Physiol 1989;11:125-34. 10. Riemer RK, Goldfien A, Roberts JM. Estrogen increases
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11.
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16. 17.
Estrogen receptors in cultured uterine myocytes
adrenergic but not cholinergic-mediated production of inositol phosphates in rabbit uterus. Mol Pharmacol1987; 32:663-8. Riemer RK, Wu YV, Bottari SP, Jacobs MM, Goldfien A, Roberts ]M. Estrogen reduces beta-adrenoceptor-mediated cAMP production and the concentration of the guanyl nucleotide-regulatory protein, G" in rabbit myometrium. Mol Pharmacol 1988;33:389-95. Nissenson R, Flouret G, Hechter O. Opposing effects of estradiol and progesterone on oxytocin receptors in rabbit uterus. Proc Nat! Acad Sci USA 1978;75:2044-8. MacKenzie LW, Garfield RE. Hormonal control of gap junctions in the myometrium. Am] PhysioI1985;17:C296308. Gilbert SF, Migeon BR. D-Valine as a selective agent for normal human and rodent epithelial cells in culture. Cell 1975;5:11-7. Taylor CM, Blanchard BB, Zava DT. A simple method to determine whole cell uptake of radiolabelled estrogen and progesterone and their subcellular localization in breast cancer cell line in monolayer culture. ] Steroid Biochem 1984;20: 1983-8. Murlas C, Nadel]A, Roberts]M. The muscarinic receptors ofaiIWay smooth muscle.] Appl PhysioI1982;52:1084-91. Kassis ]A, Walent ]H, Gorski J. Estrogen receptors in rat
18. 19. 20. 21. 22.
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uterine cell cultures: effects of medium on receptor concentration. Endocrinology 1984; 115:762-9. McCormack SA, Glasser SR. Differential response of individual uterine cell types from immature rats treated with estradiol. Endocrinology 1980; 106: 1634-49. Katzenellenbogen BS, Gorski J. Estrogen action in vitro. ] Bioi Chern 1972;247: 1299-1305. Krosl], Breskvar K, Hudnik-Plevnik T. Prolonged cultivation of rat uterine cells with preserved estrogen responsiveness.] Steroid Biochem 1989;33:189-94. Sumida C, Pasqualini JR. Estrogen responsiveness of fetal guinea pig uterine cells in culture. J Steroid Biochem 1986;24:231-4. Zaino ]R, Clarke CL, Feil PD, Satyaswaroop PG. Differential distribution of estrogen and progesterone receptors in rabbit uterus detected by dual immunofluorescence. Endocrinology 1989; 125:2728-34. Cunha GR, Young P, Brody JR. Role of uterine epithelium in the development of myometrial smooth muscles cells. Bioi Reprod 1989;40:861-71. Jacobson L, Riemer RK, GoldfienAC, Lykins D, Siiteri PK, Roberts JM. Rabbit myometrial oxytocin and a 2 -adrenergic receptors are increased by estrogen but are differentially regulated by progesterone. Endocrinology 1987; 120: 1184-9.
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