0021-972X/79/4803-0437S02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1979 by The Endocrine Society
Vol. 48, No. 3 Printed in U.S.A.
Inhibition of Human Chorionic Gonadotropin-Induced Progesterone Synthesis by Estradiol in Isolated Human Luteal Cells* MARVIN T. WILLIAMS, MICHAEL S. ROTH, JOHN M. MARSH, AND WILLIAM J. LEMAIRE
Departments of Biochemistry and Obstetrics-Gynecology and the Endocrine Laboratory, University of Miami School of Medicine, Miami, Florida 33152 ABSTRACT. The purpose of this study was to determine whether estrogens exerted a direct inhibitory effect on progesterone synthesis in isolated human luteal cells in vitro. It was found that hCG stimulated progesterone synthesis by luteal cells, obtained from corpora lutea of the menstrual cycle, whereas cells isolated from corpora lutea of pregnancy were unresponsive to exogenous hCG. Estradiol markedly inhibited (P < 0.001) this hCG effect in luteal cells of the menstrual cycle,
H
OFFMAN (1) was probably the first investigator to implicate estrogens in the luteolytic process in humans by showing that estradiol (E2) injected into the ovary caused an early onset of menstrual bleeding. More recently, Johansson and Gemzell (2), Gore et al. (3), and Vahapassi and Adlercreutz (4) showed that estriol (E3) and synthetic steroidal and nonsteroidal estrogens could advance the onset of menstruation by up to 4 days. They also showed that this probably involved a luteolytic effect as reflected by a diminished peripheral plasma progesterone concentration. Lehmann et al. (5) found the same decrease in plasma progesterone with no shortening of the luteal phase. These previously published in vivo observations could be explained by either a direct effect of the estrogens on the corpus luteum or by an. indirect effect, such as the inhibition of gonadotropin secretion or the stimulation of a uterine luteolytic factor. The purpose of our study was to determine whether estrogens could exert a direct inhibitory effect on progesterone synthesis using an in vitro system of isolated human luteal cells.
Subjects and Methods Subjects The subjects included a total of 14 women, aged 24-49 yr, who were undergoing laporotomy for various surgical proceReceived July 17,1978. Address requests for reprints to: Dr. William J. LeMaire, University of Miami, P.O. Box 016960 Miami, Florida 33101. * This work was supported in part by NIH Grants HD-03142 and HD-08747, Ford Foundation Grant 0338, and a United Way grant.
and this inhibition was dose dependent. Estradiol did not block the stimulation of cAMP accumulated by hCG in the luteal cells of the cycle but did inhibit the stimulatory effect of dibutyryl cAMP on progesterone synthesis. These data suggest that estrogens may directly cause functional luteolysis in the human and that its site of action may be after the accumulation of cAMP. (J Clin Endocrinol Metab 48: 437, 1979)
dures such as a repeat elective caesarean section; termination of pregnancy by hysterectomy; hysterectomy with and without salpingo-oophorectomy; and lysis of pelvic adhesions. All subjects signed informed consents approved by The Human Research Committee of the University of Miami School of Medicine. The age of the corpus luteum was determined from an endometrial biopsy, whenever this was carried out, or was calculated from the last menstrual period. The gestational age was calculated from the date of the last menstrual period, and pregnancy confirmed by the recovery of an embryo or fetus. Materials Type 1 collagenase CLS, 158 U/mg, lima bean trypsin inhibitor, and DNAse (2000 U/mg) were obtained from Worthington Chemical Co. Fraction V bovine serum albumin, hCG (2400 IU/mg), dibutyryl cAMP, and steroids were purchased from Sigma. The [l,2,6,7-3H]progesterone and [8-3H]cAMP were obtained from New England Nuclear. Ham's F-10 medium was purchased from Grand Island Biological Co. Tissue incubation The corpus luteum was removed intact from the ovary and immediately placed in a container on ice and transported to the laboratory. Isolated human luteal cells were prepared by a modification of the procedure of Stouffer et al. (6). The corpora lutea were sliced, minced, and incubated in air at 37 C with stirring for 1 h in the presence of 0.2% collagenase, 0.05% lima bean trypsin inhibitor, and 4 /ig DNAse/ml final incubation medium (Ham's F-10). Small remaining clumps of tissue were dissociated by drawing these clumps into a siliconized Pasteur pipette. The cell suspension was poured through a 105-jwn
437
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WILLIAMS ET AL.
438
nylon filament mesh and the filtrate was centrifuged at 100 X g for 5 min. The cells in the pellet were washed with fresh Ham's F-10 medium and counted in a standard hemocytometer. The viability was determined to be greater than 90% by the trypan blue exclusion procedure of Tennant (7). Approximately 5 X 105 cells (0.1-ml aliquot cell suspension) were incubated in air with shaking at 37 C in Ham's F-10 medium with various test substances in a final volume of 0.5 ml. The steroids were prepared in 0.5% ethanol in Ham's F-10 medium at a concentration of 100 jug/ml. Fifty microliters of this solution were added to the appropriate samples, and 50 jul 0.5% ethanol in Ham's F-10 were added to all other samples. All incubations were terminated by the addition of 55 jtd 100% trichloroacetic acid.
JCE&M • 1979 Vol 48 • No 3
pared to the variation among different corpora lutea. The data were statistically analyzed by paired comparison utilizing Student's t test; P values greater than 0.05 were not considered significant. Results There was no change in the viability (90%) of the cells after incubation, as determined by the trypan blue exclusion test (7). As shown in the upper portion of Table 1, isolated human luteal cells obtained from eight corpora lutea of the menstrual cycle responded to hCG (40 IU/ml) with a marked increase in progesterone synthesis during a 1-h incubation (P < 0.001). E2 significantly blocked this hCG-stimulated increase in progesterone synthesis (P < 0.001). Although it appears that E2 also suppressed the control level of steroidogenesis, this effect was not statistically significant. Other estrogens, estrone (E0, E 3 and diethylstilbesterol (DES) also seemed to inhibit the effect of hCG, but the number of experiments were too few to determine their statistical significance. In Exp 2129 (in the lower portion of Table 1), isolated cells from a corpus luteum obtained at 5 weeks of gestation appeared to be marginally stimulated by hCG. Moreover, E2, Ei, and DES blocked the accumulation of progesterone in this experiment. Cells isolated from five other corpora lutea obtained from later stages of pregnancy appeared to be refractory to exogenous hCG. This inhibitory effect of active estrogens was not observed with other steroids, such as 17a-E2, T, DHT, and cortisol (Table 2). In fact, DHT seemed to further stimulate progesterone accumulation in these incubated cells.
Progesterone and cAMP measurement Total progesterone (cells and medium) was determined by RIA according to a modification of the procedure of Abraham et al. (8). The modification was the measurement of progesterone directly in ether extracts of the cells and medium without purification by chromatography. The antisera (no. 337) used was a gift from Dr. G. Niswender. This antiserum had negligible cross-reactivity with 20/?Thydroxypregn4-en-3-one, testosterone, (T), and 5a-dihydrotestosterone (DHT). Intra- and interassay variability was less than 10% and the RIA data were analyzed using a computer program designed by Rodbard and Lewald (9). cAMP determinations were done using the Gilman procedure as modified by Ling et al. (10). Tracer amounts of [3H]progesterone and [3H]cAMP were added to the incubations before the extraction to correct for procedural losses. All incubations were carried out and analyzed in duplicate. The values reported in the tables are the averages of these measurements. The variation between duplicate samples was negligible com-
TABLE 1. Effect of estrogens on control and hCG-stimulated progesterone synthesis in isolated human luteal cells of menstrual cycle and pregnancy Progesterone content (ng/5 X 105cells)" Exp. no.
Age of corpus luteum
Corpora lutea of cycle 1-3 days 2144 4-6 days 2121 4-6 days 2148 5-6 days 2122 5-7 days 2090 6 days 2140 8-10 days 2125 b 2093 Corpora lutea of pregnancy 2129 5 weeks 2124 6 weeks 2112 14 weeks 16 weeks 2096 18 weeks 2113 >36 weeks 2115
Unincubated control
Incubated control
hCG
E2
hCG + E2
32.7 5.2 13.8 28.3 18.3 22.9 19.9 8.6
47.9 9.6 15.7 38.4 31.2 21.8 35.0 8.0
81.8 36.0 40.6 87.8 78.8 28.8 75.6 21.6
56.5 8.8 — 29.8 14.7 15.1 14.8 6.8
57.5 12.8 18.7 38.8 23.3 16.1 24.5 9.0
5.2 13.0 23.0 7.9 8.2 8.6
8.0 11.0 18.7 10.2 15.4 10.0
11.4 12.2 19.8 11.1 13.5 9.7
3.3 14.5 17.6 8.0 14.0 6.2
3.6 11.6 18.4 8.0 13.0 7.4
E,
hCG + E,
E3
hCG + E3
8.7
26.3
10.4
56.7
DES
hCG + DES 58.3
16.7
12.7
15.1
16.3
17.2
11.9 7.8
13.6 8.4
25.4 8.7
18.4
7.4 11.3 19.6 9.0 12.2
7.8 9.6 16.4 10.1 13.1
4.7 11.8 15.7 12.1 16.3
5.6 22.4 17.4 10.3 16.8
Isolated luteal cells were incubated in 0.5 ml Ham's F-10 medium, pH 7.4, at 37 C for 1 h. Progesterone was determined by RIA. The hCG was used at a concentration of 40 IU/ml. All steroids were used at a concentration of 10 jug/ml. ° These values are the averages of duplicate measurements. * Last menstrual period uncertain, and endometrial biopsy sample not obtained.
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ESTROGEN BLOCK OF LUTEAL STEROIDOGENESIS In Table 3 it can be seen that the inhibitory effect of E2 is dose dependent and that the minimal effective concentration is between 0.1-1.0 jug/ml. Table 4 shows that dibutyryl cAMP (2 mM) could also stimulate progesterone synthesis in isolated human luteal cells. Furthermore, E2 blocked this dibutyryl cAMP-stimulated progesterone synthesis. Although E2 markedly inhibited steroidogenesis in these isolated cells, it did not affect the control or hCG-stimulated endogenous cAMP production (Table 5).
439
jug/ml) required for inhibition of progesterone synthesis in this study appears to be rather high and possibly pharmacological, but, in view of the findings of Butler et al. (13) in the rhesus monkey, this concentration may be TABLE 5. Effect of E2 on hCG-stimulated cAMP accumulation in isolated human luteal cells of menstrual cycle cAMP content (pmol/5 X 105 cells)"
Discussion The results of this study show that estrogens can directly inhibit hCG-stimulated progesterone synthesis in isolated human luteal cells of the menstrual cycle. This observation is consistent with similar in vitro studies in the monkey (11) and the ewe (12). Therefore, it is highly conceivable that previous in vivo studies (1-4) demonstrating an inhibition of luteal function by estrogens in the human might have been due to such a direct action. The minimum dose of estrogen (between 0.1-1.0
Exp. no.
Age of corpus luteum
2140 2144 2148
6 days 1-3 days 4-6 days
Unincubated control
Incubated control
4.4
4.4
13.8
6.7
10.8 16.4
6.4
9.5 5.2
6.1
4.7
14.5
b
16.3 18.9
hCG E2
hCG
Incubations were carried out as described in Table 1. The hCG was used at a concentration of 40 IU/ml and E2 was used at a concentration of 10 jiig/ml. cAMP was measured using the procedure of Ling et al. (13). " These values are the averages of duplicate measurements. * Measurements not performed.
TABLE 2. Specificity of E2 inhibition of progesterone synthesis in isolated human luteal cells of menstrual cycle Progesterone content (ng/5 X 105 cells) Exp. no.
2140 2144
2148
Age of corpus luteum
Unincubated control
6 days 1-3 days 4-6 days
Incubated control
HCO
hCG + E2 16.1 57.5 18.7
22.9
21.8
28.8
32.7 13.8
47.9
81.8 40.6
15.7
hCG + 17aE2 30.7
h C G •+• T
hCG + DHT
hCG + cortisol
63.2
32.0
26.2 95.2
99.1
Incubations and measurements were carried out as described in Table 1. The hCG was used at a concentration of 40 IU/ml. All steroids were used at a concentration TABLE 3. Dose-related inhibition of hCG-stimulated progesterone synthesis by E2 in isolated human luteal cells of menstrual cycle Progesterone content (ng/5 X 10s cells) Exp. no. 2140 2144
Age of corpus luteum
Unincubated control
Incubated control
hCG
22.9 32.7
21.8 47.9
28.8 81.8
6 days 1-3 days
hCG + 10 jug
E 2 /ml 16.1 57.5
hCG + 1 ng E 2 /ml
hCG + 0.1 jug E 2 /ml
20.0 67.9
91.4
31.0
Incubations and measurements were carried out as described in Table 1. The hCG was used at a concentration of 40 IU/ml and E 2 was used at the indicated concentrations. TABLE 4. Effect of E2 on dibutyryl cAMP (DBcAMP)-stimulated progesterone synthesis in isolated human luteal cells of menstrual cycle and pregnancy Progesterone content (ng/5 X 105 cells) Exp. no.
Age of corpus luteum
Corpora lutea of cycle 2140 2148 Corpora lutea of pregnancy 2129
6 days 4-6 days 5 weeks
Unincubated control
Incubated control
hCG
DBcAMP
DBcAMP + E2
22.9 13.8
21.8 15.7
28.8 40.6
47.2 42.5
27.9 17.4
5.2
8.0
11.4
13.0
4.0
Incubations and measurements were carried out as described in Table 1. The hCG was used at a concentration of 40 IU/ml and E 2 was used at a concentration of 10 ju,g/ml. DBcAMP was used at a concentration of 2 mM in Exp 2140 and 2148 and at 4 mM in Exp 2129.
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440
WILLIAMS ET AL.
considered to be within the range of estrogen found in the corpus luteum of the late luteal phase. Isolated luteal cells obtained from corpora lutea of pregnancy were generally found in this study to be refractory to hQG stimulation. This is similar to the findings of reduced responsiveness in previous studies using tissue slice incubations (14, 15). The one exception to this observation was seen in Exp 2129 (Table 1). This corpus luteum was obtained from a very early pregnancy and, although it could be minimally stimulated by hCG, it appeared to be markedly suppressed by E2. In regard to the mechanism of estrogen inhibition, it appeared that E2 exerted its effect at some step beyond hCG-stimulated cAMP accumulation. This conclusion is based upon results presented in Tables 4 and 5 which showed that progesterone accumulation resulting from dibutyryl cAMP stimulation could be suppressed by E2 and that E2 does not inhibit hCG-stimulated endogenous cAMP accumulation. It has been shown by others (16) that E2 could inhibit the activity of the A5-3/?-hydroxysteroid dehydrogenase, A54-isomerase system. This might represent the actual site of estrogen inhibition of the steroidogenic pathway and would be consistent with the data presented in these experiments.
Acknowledgments The authors gratefully acknowledge the excellent technical assistance of Mr. Jorge E. Cidre and Mrs. Adalgisa Rojo. We would also like to thank Ms. Maria A. Rodrigues and Mrs. Gerty Aylward for the typing of the manuscript. We are indebted to Dr. G. Niswender for the gift of the progesterone antisera.
3. 4. 5. 6.
7. 8. 9. 10.
11.
12. 13. 14.
15.
References 1. Hoffmann, F., Untersuchungen uber die hormonale Beeinflussung der Lebensdauer des corpus Luteum in Zyklus der Frau, Geburtsh Frauenheilk 20: 1153, 1960. 2. Johansson, E. D. B., and C. Gemzell, Plasma levels of progesterone
16.
JCE&M • 1979 Vol 48 • No 3
during the luteal phase in normal women treated with synthetic oestrogens (RS 2874, F 6103 and ethinyloestradiol), Acta Endocrinol (Kbh) 68: 551, 1971. Gore, B. Z., B. V. Caldwell, and L. Speroff, Estrogen-induced human luteolysis, J Clin Endocrinol Metab 36: 615, 1973. Vahapassi, J., and H. Adlercreutz, Effect of continuous oral administration of estriol on corpus luteum function, Contraception 11: 427, 1975. Lehmann, F., I. Just-Nastansky, B. Behrendt, P.-J. Czygan, and G. Bettendorf, Effect of post-ovulatory administered oestrogens on corpus luteum function, Acta Endocrinol (Kbh) 79: 329, 1975. Stouffer, R. L., W. E. Nixon, B. J. Gulyas, D. K. Johnson, and G. D. Hodgen, In vitro evaluation of corpus luteum function of cycling and pregnant rhesus monkeys: progesterone production by dispersed luteal cells, Steroids 27: 543, 1976. Tennant, J. R., Evaluation of the trypan blue techniques for determination of cell viability, Transplantation 2: 685, 1964. Abraham, G. E., R. Swerdloff, D. Tulchinsky, and W. D. Odell, Radioimmunoassay of plasma progesterone, J Clin Endocrinol Metab 32: 619, 1971. Rodbard, D., and J. E. Lewald, Computer analyses of radioligand assay and radioimmunoassay data, Acta Endocrinol (Suppl) 147: 79, 1970. Ling, W. Y., J. M. Marsh, W. N. Spellacy, A. J. Thresher, and W. J. LeMaire, Adenosine 3',5'-monophosphate in amniotic fluid from pregnancy complicated by hypertension, J Clin Endocrinol Metab 39:^479, 1974. Stouffer, R. L., W. E. Nixon, B. J. Gulyas, and G. D. Hodgen, Gonadotropin-sensitive progesterone production by the rhesus monkey luteal cells in vitro: a function of the age of the corpus luteum during the menstrual cycle, Endocrinology 100: 506, 1977. Akbar, A. M., F. Stormshak, and D. J. Lee, Estradiol inhibition of ovine luteal progesterone synthesis in vitro, J Anim Sci 35: 1206, 1972. Butler, W. R., J. Hotchkiss, and E. Knobil, Functional luteolysis in the rhesus monkey: ovarian estrogen and progesterone during the luteal phase of the menstrual cycle, Endocrinology, 96: 1509,1975. LeMaire, W. J., B. F. Rice, and K. Savard, Steroid hormone formation in the human ovary. V. Synthesis of progesterone in vitro in corpora lutea during the reproductive cycle, J Clin Endocrinol Metab 28: 1249, 1968. Marsh, J. M., and W. J. LeMaire, Cyclic AMP accumulation and steroidogenesis in the human corpus luteum: effect of gonadotropins and prostaglandins, J Clin Endocrinol Metab 38: 99, 1974. Depp, R., D. W. Cox, R. J. Pion, S. H. Conrad, and W. L. Heinrichs, Inhibition of the pregnenolone A5-3/?-hydroxysteroid dehydrogenase-A5"4 isomerase systems of human placenta and corpus luteum of pregnancy, Gynecol Invest 4: 106, 1973.
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Vol. 48, No. 3 Printed in U.S.A.
Inhibition of Human Chorionic Gonadotropin-Induced Progesterone Synthesis by Estradiol in Isolated Human Luteal Cells* MARVIN T. WILLIAMS, MICHAEL S. ROTH, JOHN M. MARSH, AND WILLIAM J. LEMAIRE
Departments of Biochemistry and Obstetrics-Gynecology and the Endocrine Laboratory, University of Miami School of Medicine, Miami, Florida 33152 ABSTRACT. The purpose of this study was to determine whether estrogens exerted a direct inhibitory effect on progesterone synthesis in isolated human luteal cells in vitro. It was found that hCG stimulated progesterone synthesis by luteal cells, obtained from corpora lutea of the menstrual cycle, whereas cells isolated from corpora lutea of pregnancy were unresponsive to exogenous hCG. Estradiol markedly inhibited (P < 0.001) this hCG effect in luteal cells of the menstrual cycle,
H
OFFMAN (1) was probably the first investigator to implicate estrogens in the luteolytic process in humans by showing that estradiol (E2) injected into the ovary caused an early onset of menstrual bleeding. More recently, Johansson and Gemzell (2), Gore et al. (3), and Vahapassi and Adlercreutz (4) showed that estriol (E3) and synthetic steroidal and nonsteroidal estrogens could advance the onset of menstruation by up to 4 days. They also showed that this probably involved a luteolytic effect as reflected by a diminished peripheral plasma progesterone concentration. Lehmann et al. (5) found the same decrease in plasma progesterone with no shortening of the luteal phase. These previously published in vivo observations could be explained by either a direct effect of the estrogens on the corpus luteum or by an. indirect effect, such as the inhibition of gonadotropin secretion or the stimulation of a uterine luteolytic factor. The purpose of our study was to determine whether estrogens could exert a direct inhibitory effect on progesterone synthesis using an in vitro system of isolated human luteal cells.
Subjects and Methods Subjects The subjects included a total of 14 women, aged 24-49 yr, who were undergoing laporotomy for various surgical proceReceived July 17,1978. Address requests for reprints to: Dr. William J. LeMaire, University of Miami, P.O. Box 016960 Miami, Florida 33101. * This work was supported in part by NIH Grants HD-03142 and HD-08747, Ford Foundation Grant 0338, and a United Way grant.
and this inhibition was dose dependent. Estradiol did not block the stimulation of cAMP accumulated by hCG in the luteal cells of the cycle but did inhibit the stimulatory effect of dibutyryl cAMP on progesterone synthesis. These data suggest that estrogens may directly cause functional luteolysis in the human and that its site of action may be after the accumulation of cAMP. (J Clin Endocrinol Metab 48: 437, 1979)
dures such as a repeat elective caesarean section; termination of pregnancy by hysterectomy; hysterectomy with and without salpingo-oophorectomy; and lysis of pelvic adhesions. All subjects signed informed consents approved by The Human Research Committee of the University of Miami School of Medicine. The age of the corpus luteum was determined from an endometrial biopsy, whenever this was carried out, or was calculated from the last menstrual period. The gestational age was calculated from the date of the last menstrual period, and pregnancy confirmed by the recovery of an embryo or fetus. Materials Type 1 collagenase CLS, 158 U/mg, lima bean trypsin inhibitor, and DNAse (2000 U/mg) were obtained from Worthington Chemical Co. Fraction V bovine serum albumin, hCG (2400 IU/mg), dibutyryl cAMP, and steroids were purchased from Sigma. The [l,2,6,7-3H]progesterone and [8-3H]cAMP were obtained from New England Nuclear. Ham's F-10 medium was purchased from Grand Island Biological Co. Tissue incubation The corpus luteum was removed intact from the ovary and immediately placed in a container on ice and transported to the laboratory. Isolated human luteal cells were prepared by a modification of the procedure of Stouffer et al. (6). The corpora lutea were sliced, minced, and incubated in air at 37 C with stirring for 1 h in the presence of 0.2% collagenase, 0.05% lima bean trypsin inhibitor, and 4 /ig DNAse/ml final incubation medium (Ham's F-10). Small remaining clumps of tissue were dissociated by drawing these clumps into a siliconized Pasteur pipette. The cell suspension was poured through a 105-jwn
437
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WILLIAMS ET AL.
438
nylon filament mesh and the filtrate was centrifuged at 100 X g for 5 min. The cells in the pellet were washed with fresh Ham's F-10 medium and counted in a standard hemocytometer. The viability was determined to be greater than 90% by the trypan blue exclusion procedure of Tennant (7). Approximately 5 X 105 cells (0.1-ml aliquot cell suspension) were incubated in air with shaking at 37 C in Ham's F-10 medium with various test substances in a final volume of 0.5 ml. The steroids were prepared in 0.5% ethanol in Ham's F-10 medium at a concentration of 100 jug/ml. Fifty microliters of this solution were added to the appropriate samples, and 50 jul 0.5% ethanol in Ham's F-10 were added to all other samples. All incubations were terminated by the addition of 55 jtd 100% trichloroacetic acid.
JCE&M • 1979 Vol 48 • No 3
pared to the variation among different corpora lutea. The data were statistically analyzed by paired comparison utilizing Student's t test; P values greater than 0.05 were not considered significant. Results There was no change in the viability (90%) of the cells after incubation, as determined by the trypan blue exclusion test (7). As shown in the upper portion of Table 1, isolated human luteal cells obtained from eight corpora lutea of the menstrual cycle responded to hCG (40 IU/ml) with a marked increase in progesterone synthesis during a 1-h incubation (P < 0.001). E2 significantly blocked this hCG-stimulated increase in progesterone synthesis (P < 0.001). Although it appears that E2 also suppressed the control level of steroidogenesis, this effect was not statistically significant. Other estrogens, estrone (E0, E 3 and diethylstilbesterol (DES) also seemed to inhibit the effect of hCG, but the number of experiments were too few to determine their statistical significance. In Exp 2129 (in the lower portion of Table 1), isolated cells from a corpus luteum obtained at 5 weeks of gestation appeared to be marginally stimulated by hCG. Moreover, E2, Ei, and DES blocked the accumulation of progesterone in this experiment. Cells isolated from five other corpora lutea obtained from later stages of pregnancy appeared to be refractory to exogenous hCG. This inhibitory effect of active estrogens was not observed with other steroids, such as 17a-E2, T, DHT, and cortisol (Table 2). In fact, DHT seemed to further stimulate progesterone accumulation in these incubated cells.
Progesterone and cAMP measurement Total progesterone (cells and medium) was determined by RIA according to a modification of the procedure of Abraham et al. (8). The modification was the measurement of progesterone directly in ether extracts of the cells and medium without purification by chromatography. The antisera (no. 337) used was a gift from Dr. G. Niswender. This antiserum had negligible cross-reactivity with 20/?Thydroxypregn4-en-3-one, testosterone, (T), and 5a-dihydrotestosterone (DHT). Intra- and interassay variability was less than 10% and the RIA data were analyzed using a computer program designed by Rodbard and Lewald (9). cAMP determinations were done using the Gilman procedure as modified by Ling et al. (10). Tracer amounts of [3H]progesterone and [3H]cAMP were added to the incubations before the extraction to correct for procedural losses. All incubations were carried out and analyzed in duplicate. The values reported in the tables are the averages of these measurements. The variation between duplicate samples was negligible com-
TABLE 1. Effect of estrogens on control and hCG-stimulated progesterone synthesis in isolated human luteal cells of menstrual cycle and pregnancy Progesterone content (ng/5 X 105cells)" Exp. no.
Age of corpus luteum
Corpora lutea of cycle 1-3 days 2144 4-6 days 2121 4-6 days 2148 5-6 days 2122 5-7 days 2090 6 days 2140 8-10 days 2125 b 2093 Corpora lutea of pregnancy 2129 5 weeks 2124 6 weeks 2112 14 weeks 16 weeks 2096 18 weeks 2113 >36 weeks 2115
Unincubated control
Incubated control
hCG
E2
hCG + E2
32.7 5.2 13.8 28.3 18.3 22.9 19.9 8.6
47.9 9.6 15.7 38.4 31.2 21.8 35.0 8.0
81.8 36.0 40.6 87.8 78.8 28.8 75.6 21.6
56.5 8.8 — 29.8 14.7 15.1 14.8 6.8
57.5 12.8 18.7 38.8 23.3 16.1 24.5 9.0
5.2 13.0 23.0 7.9 8.2 8.6
8.0 11.0 18.7 10.2 15.4 10.0
11.4 12.2 19.8 11.1 13.5 9.7
3.3 14.5 17.6 8.0 14.0 6.2
3.6 11.6 18.4 8.0 13.0 7.4
E,
hCG + E,
E3
hCG + E3
8.7
26.3
10.4
56.7
DES
hCG + DES 58.3
16.7
12.7
15.1
16.3
17.2
11.9 7.8
13.6 8.4
25.4 8.7
18.4
7.4 11.3 19.6 9.0 12.2
7.8 9.6 16.4 10.1 13.1
4.7 11.8 15.7 12.1 16.3
5.6 22.4 17.4 10.3 16.8
Isolated luteal cells were incubated in 0.5 ml Ham's F-10 medium, pH 7.4, at 37 C for 1 h. Progesterone was determined by RIA. The hCG was used at a concentration of 40 IU/ml. All steroids were used at a concentration of 10 jug/ml. ° These values are the averages of duplicate measurements. * Last menstrual period uncertain, and endometrial biopsy sample not obtained.
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ESTROGEN BLOCK OF LUTEAL STEROIDOGENESIS In Table 3 it can be seen that the inhibitory effect of E2 is dose dependent and that the minimal effective concentration is between 0.1-1.0 jug/ml. Table 4 shows that dibutyryl cAMP (2 mM) could also stimulate progesterone synthesis in isolated human luteal cells. Furthermore, E2 blocked this dibutyryl cAMP-stimulated progesterone synthesis. Although E2 markedly inhibited steroidogenesis in these isolated cells, it did not affect the control or hCG-stimulated endogenous cAMP production (Table 5).
439
jug/ml) required for inhibition of progesterone synthesis in this study appears to be rather high and possibly pharmacological, but, in view of the findings of Butler et al. (13) in the rhesus monkey, this concentration may be TABLE 5. Effect of E2 on hCG-stimulated cAMP accumulation in isolated human luteal cells of menstrual cycle cAMP content (pmol/5 X 105 cells)"
Discussion The results of this study show that estrogens can directly inhibit hCG-stimulated progesterone synthesis in isolated human luteal cells of the menstrual cycle. This observation is consistent with similar in vitro studies in the monkey (11) and the ewe (12). Therefore, it is highly conceivable that previous in vivo studies (1-4) demonstrating an inhibition of luteal function by estrogens in the human might have been due to such a direct action. The minimum dose of estrogen (between 0.1-1.0
Exp. no.
Age of corpus luteum
2140 2144 2148
6 days 1-3 days 4-6 days
Unincubated control
Incubated control
4.4
4.4
13.8
6.7
10.8 16.4
6.4
9.5 5.2
6.1
4.7
14.5
b
16.3 18.9
hCG E2
hCG
Incubations were carried out as described in Table 1. The hCG was used at a concentration of 40 IU/ml and E2 was used at a concentration of 10 jiig/ml. cAMP was measured using the procedure of Ling et al. (13). " These values are the averages of duplicate measurements. * Measurements not performed.
TABLE 2. Specificity of E2 inhibition of progesterone synthesis in isolated human luteal cells of menstrual cycle Progesterone content (ng/5 X 105 cells) Exp. no.
2140 2144
2148
Age of corpus luteum
Unincubated control
6 days 1-3 days 4-6 days
Incubated control
HCO
hCG + E2 16.1 57.5 18.7
22.9
21.8
28.8
32.7 13.8
47.9
81.8 40.6
15.7
hCG + 17aE2 30.7
h C G •+• T
hCG + DHT
hCG + cortisol
63.2
32.0
26.2 95.2
99.1
Incubations and measurements were carried out as described in Table 1. The hCG was used at a concentration of 40 IU/ml. All steroids were used at a concentration TABLE 3. Dose-related inhibition of hCG-stimulated progesterone synthesis by E2 in isolated human luteal cells of menstrual cycle Progesterone content (ng/5 X 10s cells) Exp. no. 2140 2144
Age of corpus luteum
Unincubated control
Incubated control
hCG
22.9 32.7
21.8 47.9
28.8 81.8
6 days 1-3 days
hCG + 10 jug
E 2 /ml 16.1 57.5
hCG + 1 ng E 2 /ml
hCG + 0.1 jug E 2 /ml
20.0 67.9
91.4
31.0
Incubations and measurements were carried out as described in Table 1. The hCG was used at a concentration of 40 IU/ml and E 2 was used at the indicated concentrations. TABLE 4. Effect of E2 on dibutyryl cAMP (DBcAMP)-stimulated progesterone synthesis in isolated human luteal cells of menstrual cycle and pregnancy Progesterone content (ng/5 X 105 cells) Exp. no.
Age of corpus luteum
Corpora lutea of cycle 2140 2148 Corpora lutea of pregnancy 2129
6 days 4-6 days 5 weeks
Unincubated control
Incubated control
hCG
DBcAMP
DBcAMP + E2
22.9 13.8
21.8 15.7
28.8 40.6
47.2 42.5
27.9 17.4
5.2
8.0
11.4
13.0
4.0
Incubations and measurements were carried out as described in Table 1. The hCG was used at a concentration of 40 IU/ml and E 2 was used at a concentration of 10 ju,g/ml. DBcAMP was used at a concentration of 2 mM in Exp 2140 and 2148 and at 4 mM in Exp 2129.
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440
WILLIAMS ET AL.
considered to be within the range of estrogen found in the corpus luteum of the late luteal phase. Isolated luteal cells obtained from corpora lutea of pregnancy were generally found in this study to be refractory to hQG stimulation. This is similar to the findings of reduced responsiveness in previous studies using tissue slice incubations (14, 15). The one exception to this observation was seen in Exp 2129 (Table 1). This corpus luteum was obtained from a very early pregnancy and, although it could be minimally stimulated by hCG, it appeared to be markedly suppressed by E2. In regard to the mechanism of estrogen inhibition, it appeared that E2 exerted its effect at some step beyond hCG-stimulated cAMP accumulation. This conclusion is based upon results presented in Tables 4 and 5 which showed that progesterone accumulation resulting from dibutyryl cAMP stimulation could be suppressed by E2 and that E2 does not inhibit hCG-stimulated endogenous cAMP accumulation. It has been shown by others (16) that E2 could inhibit the activity of the A5-3/?-hydroxysteroid dehydrogenase, A54-isomerase system. This might represent the actual site of estrogen inhibition of the steroidogenic pathway and would be consistent with the data presented in these experiments.
Acknowledgments The authors gratefully acknowledge the excellent technical assistance of Mr. Jorge E. Cidre and Mrs. Adalgisa Rojo. We would also like to thank Ms. Maria A. Rodrigues and Mrs. Gerty Aylward for the typing of the manuscript. We are indebted to Dr. G. Niswender for the gift of the progesterone antisera.
3. 4. 5. 6.
7. 8. 9. 10.
11.
12. 13. 14.
15.
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JCE&M • 1979 Vol 48 • No 3
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