- Email: [email protected]
Inhibition of induction of 20α-hydroxysteroid dehydrogenase activity in rat corpora lutea in vitro by the progesterone antagonist RU486
Life Sciences, Vol. Printed in the USA
50, pp.
1173-1178
Pergamon
Press
INHIBITION OF INDUCTION OF 20g-HYDROXYSTEROID DEHYDROGENASE ACTIVITY IN RAT CORPORA LUTEA IN VITRO BY THE PROGESTERONE ANTAGONIST RU486 J.Th.J. Uilenbroek, P.J.A. Woutersen and B. Karels Department of Endocrinology and Reproduction, Faculty of Medicine, Erasmus University Rotterdam The Netherlands (Received
in final
form February
I0,
1992)
Summary To determine if the antiprogestagen RU486 has a direct effect on luteal progesterone secretion, whole corpora lutea or dispersed luteal cells were incubated in the presence of RU486. Whole corpora lutea, isolated from rats at day 5 of pseudopregnancy, were incubated individually in hormone-free medium. The concentrations of progesterone and 20n-dihydroprogesterone in the medium plus corpus luteum was measured before and after 24 h of incubation. In the absence of RU486 the concentration of 20~-dihydro-progesterone increased, while that of progesterone remained unchanged. In the presence of RU486 (230 I.tM) the concentration of both progesterone and 20a-dihydro-progesterone was increased. Dispersed luteal cells were incubated for 24 h in the presence of various amounts of RU486. In the absence and in the presence of 0.2 and 2.3 I.tM RU486 a high ratio between 20~dihydro-progesterone and progesterone was found, while in the presence of 23 ~M RU486 the concentrations of progesterone and 20c~-dihydro-progesterone were equal. 20~-Hydroxysteroid dehydrogenase (20~-HSD) activity measured in luteal homogenates started to increase between 6 and 12 h of incubation. This increase could be prevented after incubation of the corpora lutea in the presence of 23 or 230 I.tM RU486 for 24 hrs. It is concluded that the progesterone antagonist RU486 can have a direct effect on luteal progesterone production. RU486 prevents the increase in 20g-HSD activity that normally occurs during in vitro incubation. However, since these effects in vitro can only be obtained with high concentrations of RU486, it is unlikely that this antiluteolytic effect plays a role after injection of RU486 in vivo.
RU486 (•7B-hydr•xy-••B-[4-dimethy•-amin•pheny•]-•7a•[•-pr•pyny•]•estra-4•9-diene-3-•ne) is a synthetic 19-norsteroid that binds to the progesterone receptor with high affinity and acts as a progesterone antagonist (1). In humans and non-human primates it induces early menstruation when given during the luteal phase of the menstrual cycle (2,3) and abortion when given during early pregnancy (4,5). This effect is due to inhibition of receptor-mediated progesterone action on the uterus. However, progesterone receptors are also present in the ovary (6) and RU486 binds to this receptor with high affinity (7). Luteolytic and antiluteolytic effects of RU486 have been described (8,9). Most of these effects could be attributed to changes in circulating levels of prolactin. However, it has been hypothesized that progesterone also plays a role in the regulation of its own production (10). Therefore it is the objective of the present study to investigate whether RU486 might have a direct effect on luteal progesterone production. A possible direct effect of RU486 on the corpus luteum in vivo is difficult to assess as progesterone influences the secretion of gonadotrophins and prostaglandins, which are required for normal luteal function (10). Therefore the effect of RU486 on luteal activity has been studied in vitro. Correspondence should be addressed to Dr. J. Uilenbroek
Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press plc All rights reserved.
1174
RU486 and Corpus Luteum Activity
Vol.
50, No. 16, 1992
Materials and Methods Animals Adult Wistar rats (3-5 months old) were used in this study. The animals were housed under standard conditions of light (on: 05.00-19.00 h) and temperature (20-23°C). The females showed almost exclusively 5-day reproductive cycles as detected by vaginal smears, which were made 5 days a week. The animals were made pseudopregnant by caging them overnight with a vasectomized male. At day 5 of pseudopregnancy (the day of estrus was considered as the first day of pseudopregnancy) the animals were sacrificed, the ovaries were dissected out and the last generation of corpora lutea was isolated under a dissection microscope. The corpora lutea were either incubated intact or collected in Dulbecco's phosphate buffered saline (PBS) for the preparation of dispersed luteal cells. Incubation of whole corpora lutea Whole corpora lutea were incubated individually in 1 ml Medium-199 (Gibco, Grand Island, NY, USA), pH 7.4, containing Hepes (25 mM) (Sigma Chemical Co., St. Louis, MO, USA), antibiotic-antimycotic mixture (250 p.g amphotericin, 105 IU penicillin, 100 mg streptomycin sulfate per 1) (Gibco, Grand Island, NY, USA) and various amounts of RU486 (RousseI-Uclaf, France) dissolved in 10 ~1 ethanol. The incubations were carried out at 37°C under an atmosphere of 95% 02 and 5% CO 2 in a shaking waterbath. At the end of the incubation 1 ml ethanol was added to the medium and the corpus luteum was crushed. The mixture was stored at -20°C until measurement of the progesterone and 20a-dihydro-progesterone content. Dispersion of luteal cells Isolated corpora lutea from 3 to 4 rats (12-14 corpora lutea per rat) were washed in Dulbecco's PBS, pooled and incubated in 5 ml Medium Essential Medium (MEM, Gibco, Grand Island, NY, USA) containing 0 . 1 % collagenase, 0.02 % DNAse and 1 % fetal calf serum (FCS). Collagenase, DNAse and FCS were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The corpora lutea were incubated for two consecutive 30 min periods in a shaking waterbath at 37°C. After each incubation the corpora lutea were mechanically dispersed with a plastic Pasteur pipet. After the last incubation, tissue fragments were removed by filtration through cheesecloth. The dispersed cells wcrc collected by centrifugation (5 min, 800 g) and the pellet was washed three times with MEM containing 1% FCS. The pellet was resuspended in MEM containing 1% FCS in a final concentration of 1 corpus luteum per 100 ILl. The dispersed cells equivalent to 1 corpus luteum were incubated in 1 ml MEM containing 1% FCS and increasing amounts of RU486 (0.23 - 23 nmol) at 37°C in an humidified atmosphere of 5% CO 2 and 95% air. After incubation for 24 h the incubation medium was centrifuged (10 min, 2500g) and the supernatant was stored at -20°C for assay of progesterone and 20ndihydro-progesterone. Steroid assays The incubation medium containing the crushed corpora lutea was centrifuged for 10 min at 2500 g and the pellet was extracted twice with 500 l.tl acetone. The combined supernatants were evaporated to dryness and redissolved in 1 ml PBS (0.01 M, pH 7.0) containing 0.15% (w/v) gelatin. Progesterone and 20e-dihydroprogesterone concentrations were measured by radio-immunoassay as described previously (11). Since high amounts of RU486 were used in the incubation medium special attention was given to the validation of steroid measurements. Crossreactivity of RU486 with the progesterone and 20~-dihydroprogesterone antibodies was less than 0.01%. In a number of incubation samples containing RU486 (230 ~M), progesterone was measured with and without chomatography. The samples were extracted twice with hexaneaether (4:1, v/v). Thin layer chromatography (TLC) was performed using precoated silica-plates G F ~ (Merck, Darmstadt, Germany) as stationary phase and chloroform-acetone (95:5, v/v) as mobile phase. As samples measured with TLC were not lower than those measured without TLC, progesterone and 20n-dihydroprogesterone measurements in the presence of RU486 (230 p.M) were considered to be reliable. Enzyme assays For the measurement of 20ct-hydroxysteroid dehydrogenase (20ct-HSD) activity corpora lutea were weighed, homogenized in 0.05 M Trls/EDTA-buffered saline pH 7.4 and centrifuged at 800 g (5 min 4°C). One hundred I-d supcrnatant of a 3% homogenate was incubated with (4 x 104 d.p.m) [1,2,6,7,16,17 3H] progesterone (s.a. 140 C/mmol), 16 nmol progesterone, 500 I.tl of a NADPH-regenerating system (containing 1.4 U glucose 6-phosphate dehydrogenase, 20 mM glucose 6-phosphate and 2raM NADP) and 250 p.l of an inhibitor of aromatase activity (androsta-l.4.6-tricne-3.17-dione, ATD, 400 p.M) and an inhibitor of 5ctreductase (4-androsten-3-onc-17B carboxylic acid, AC, 398 p.M) in a final concentration of 1 ml Tris/EDTA.
Vol.
50, NO.
16, 1992
RU486 and Corpus Luteum Activity
1175
After incubation for 30 min in a shaking waterbath at 37°C the reaction was stopped by adding 1.5 ml ice-cold hexane-aether (4:1 v/v). After extraction twice with hexane-aether progesterone and 20=-dihydro-progesterone were separated by TLC as described above. The enzyme activity was expressed in pmol substrate converted per min per mg luteal tissue. 3H-labeled and unlabeled steroids used were obtained from Amersham International PLC (Amersham, UK). To investigate whether RU486 had a direct antagonistic effect on the 20n-HSD activity the enzyme activity was also measured in the presence of RU486. Corpora lutea were first incubated for 24 hrs in the absence of RU486. Thereafter a 3% luteal homogenate was incubated with increasing amounts of 3H-labeled progesterone and unlabeled progesterone as substrate in the presence and absence of 12 nmol RU486. Statistics Results are given as means ± S.E.M. Statistical analysis consisted of Student's t-test and one-way analysis of variance (ANOVA). If the overall test was significant, comparison between groups was made by the Least Significant Difference (LSD) test. A difference was considered significant if the probability (P) was <0.05 (two-tailed). Results Effect of RU486 on whole corpora lutea and dispersed luteal cells in vitro At day 5 of pseudopregnancy corpora lutea contained 0.27 ± 0.02 nmol progesterone and 0.13 ± 0.03 nmol 20ct-dihydro-progesterone per corpus luteum. After 24 h of incubation the concentration of progesterone in the corpus luteum plus medium remained constant (0.26 ± 0.02 nmol), while that of 20ct-dihydroprogesterone increased to 1.04 ± 0.1 nmol (P<0.01, Student's t-test). Incubation in the presence of RU486 (230 I.tM) resulted in high concentrations of both progesterone and 20et-dihydro-progesterone (1.30 ± 0.17 nmol and 1.45 ± 0.16 nmol respectively). Incubation of dispersed luteal cells in the absence and presence of 0.2 and 2.3 I~M RU486 for 24 h resulted in a low concentration of progesterone and a high concentration of 20ct-dihydro-progesterone in the medium (Fig. 1). However, incubation in the presence of 23 I.tM RU486 resulted in a significantly higher concentration of progesterone and a lower concentration of 20et-dihydro-progesterone (ANOVA; LSD=7.79 pmol for progesterone and 47.7 pmol for 20ct-dihydro-progesterone).
300
i
\\
200
\\\
:
\\\ \\\
\\
---e---
\
prog 20a-OHprog
100 o
0 0.1
1
10
100
RU48S ( . M )
Fig. 1. Concentrations of extracellular levels of progesterone (e) and 20¢t-dihydro-progesterone (o) after incubation of dispersed luteal cells for 24 h in the presence of increasing amounts of RU486. The data represent the mean ± S.E.M. of 4 incubations. When error bars are absent, they are too small to indicate.
1176
RU486 and Corpus Luteum Activity
Vol. 50, No. 16, 1992
Effect of RU486 on 20a-HSD activity The activity of 20a-HSD in homogenized corpora lutea was measured before and after 3, 6, 12, 24 and 48 h of incubation. Before incubation the activity was low (< 3 pmol/min/mg luteal tissue). Between 6 and 12 h of incubation the activity increased significantly (ANOVA; LSD=5.34 pmol) and was high after 24 and 48 h of incubation (Fig. 2). Incubation in the presence of RU486 for 24 h prevented the increase of 20a-HSD activity in a dose dependent manner (Table I). A concentrations of 2.3 I~M was already effective (ANOVA; LSD =3.50 pmol).
100 •
E
80
T
m
E 0
E
60
40 .I-
6
0
20
04
0 0
i
,
,
i
12
24
36
48
hrs of Incubation
Fig. 2. 20a-hydroxysteroid dehydrogenase (20a-HSD) activity in corpora lutea before and after incubation. The activity is measured in a 3% homogenate and is expressed as pmol/min/mg luteal tissue. The data represent the mean ± S.E.M. of triplicate measurements. When error bars are absent, they are too small to indicate.
TABLE 1 20a-Hydroxysteroid dehydrogenase (20a-HSD) activity in whole corpora lutea after 24 h of incubation in the absence and presence of RU486. The values represent the mean ± S.D. of duplicate measurements. RU486 (p.M)
-2.3 23 230
20a-HSD activity (pmol/min/mg)
46.4 ± 0.9 35.9 ± 1.6 14.6 ± 0.0 7.6 ± 0.6
Vol. 50, No. 16, 1992
RU486 and Corpus Luteum Activity
1177
To investigate whether the low 20a-HSD activity found after incubation with RU486 might be due to an inhibition of the induction of this enzyme or to a direct competitive inhibition of the conversion of progesterone to 20ct-dihydro-progesterone the following experiment was performed. Corpora lutea were first incubated for 24 h to ensure a high 20rt-HSD activity. Thereafter 20tz-HSD activity was measured in the presence and absence of RU486 (12 I~M). As the conversion rate between progesterone to 20--OH progesterone was not different between the two groups, it was concluded that the low 20~-HSD activity after 24 h of incubation in the presence of RU486 was not due to competitive inhibition of RU486 with the snbstrate progesterone on the enzyme. Discussion Corpora lutea of pseudopregnant rats secrete large amounts of progesterone. During luteolysis at the end of pseudopregnancy progesterone in the circulation decreases. This decrease is partially due to a decrease in progesterone production, but mainly to the conversion of progesterone to the biologically inactive metabolite 20n-dihydro-progesterone by the microsomal enzyme 20~t-HSD (12). The present study shows that the activity of this enzyme in corpora lutea increases during incubation in hormone-free medium, resulting in a low concentration of progesterone and a high concentration of 20adihydro-progesterone at the end of the incubation period. The increase starts after 6 h suggesting "de novo" synthesis. The mechanism by which 20¢t-HSD activity increases during incubation is not know. In vivo the activity is low in the presence of high prolactin levels (13) and increases as prolactin declines (14). From these data it is conceivable that the increase in vitro is due to the absence of prolactin in the culture medium. However, addition of prolactin to the culture medium did not prevent the increase in 20a-HSD activity (Uilenbroek, unpublished results), although such an effect has been reported by others (15). The present data show that the increase in 20ct-HSD activity is prevented by addition of RU486 to the culture medium. The low enzyme activity at the end of the incubation period with RU486 is not due to a competitive inhibition of the compound with progesterone on the enzyme. Rather the antiprogestagen inhibits the synthesis of this enzyme. From the inhibitory action of RU486 on the increase of 20tz-HSD activity it can be concluded that the increase in 20a-HSD activity in vitro is a progesterone-mediated event. An effect of progesterone on its own activity has been proposed by Rothchild (10). However, this author concluded that progesterone stimulates and does not inhibit its own production. The effect of RU486 on 20a-HSD activity can only be obtained with high concentrations of RU486. Large amounts might be required to block a possible autocrine effect of progesterone on its own metabolism. Furthermore, these high levels might be of relevance for in studies performed in vivo, adult rats were given daily injections with 2 mg/day. Assuming an equal distribution and no rapid metabolism corpora lutea in vivo are then exposed to I~M concentrations of RU486. An inhibition of 20~t-HSD activity by RU486 should result in an increased concentration of progesterone and a decreased concentration of 20ct-dihydro-progesterone. This was found in the medium after incubation of luteal cells with 23 o.M RU486. Incubation with whole corpora lutea however, resulted in high concentrations of both progesterone and 20a-dihydro-progesterone, while an increase in 20a-HSD activity was already prevented at 2.3 I~M RU486. This discrepancy between enzyme activity and the content of progesterone and 20tz-dihydro-progesterone in the whole corpora lutea might be related to the lower penetration rate of RU486 in the whole corpora lutea compared with dispersed cells. It is likely that in whole copora lutea a considerable amount of 20ct-dihydro-progesterone has already been formed before a further increase of 20ct-HSD activity occurs. A direct effect of RU486 on human ovarian steroidogenesis has been reported by Dimattina et al. (16,17). They found an inhibition of progesterone production by human granulosa cells due to inhibition of 3BHSD activity (16). Furthermore, a decrease in 17-hydroxylase activity with doses RU486 exceding 50 ~tM has also been reported (17). A direct effect of RU486 on LH receptor binding or cyclic AMP generation using a membrane preparation from human corpora lutea has not been found (18). A direct antiluteolytic effect of RU486 in vitro is in line with some results observed in vivo. Daily injections of RU486 (2 mg/rat/day) for 28 days result in an increase of serum progesterone levels (9). These high levels of progesterone however, are due to the high circulating levels of prolactin (19). Moreover, in lactating rats RU486 did not increase luteal progesterone production (20). In lactacting rats prolactin levels are high due to the suckling stimulus and are not different between RU486 treated rats and controls. So in vivo studies do not support a direct effect of RU486 on luteal progesterone production.
1178
RU486
and C o r p u s L u t e u m A c t i v i t y
Vol.
50, No.
16, 1992
In conclusion, incubation of corpora lutea with large amounts of RU486 results in an inhibition of the conversion of progesterone to 20~x-OH progesterone suggesting that progesterone itself might play a role in the increase of 20n-HSD activity. However, I.tM concentrations are required to obtain these effects. It is therefore unlikely that the effects found in vitro play a major role in the effects observed after injections of RU486 in vivo. Acknowledgement The authors are indebted to Dr. R. Deraedt (Roussel-Uclaf, France) for supplying RU486. References
2,
3. 4.
9.
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
D. PHILIBERT, M. MOGUILEWSKY, I. MARY, D. LECAQUE, C. TOURNEMINE, J. SECCHI and R. DERAEDT, The antiprogestin steroid RU486 and human fertility control, Baulieu E.E. and Segal S. (eds), 49-68, Plenum Press, New York, (1985). D.L. HEALY, E.E. BAULIEU and G.D. HOGDEN, Fertility and Sterility 40 253-257 (1983). B. SHORTLE, I. DYRENFURTH and M. FERIN, J Clin Endocrinol Metab 60 731-735 (1985). L. KOVACS, M. SAS, B.A. RESCH, G. UGOSCA1, M.L. SWAHN, M. BYGDEMAN and P.J. ROWE, Contraception 29 399-410 (1984). B. COUZINET, N. LE STRAT, A. ULMANN, E.E. BAULIEU and G. SCHAISON, New England Journal of Medicine 3!5 1565-1570 (1986). J.R. SCHREIBER and A.J.W. HSUEH, Endocrinology 10__._55915-919 (1979). J.R. SCHREIBER, AJ.W. HSUEH and E.E. BAULIEU, Contraception 28 77-85 (1983). T. KAWANO, H. OKAMURA, C. TAJIMA, K. FUKUMA and H. KATABUCHI, J Reprod Fert 83 279-285 (1988). P. VAN DER SCHOOT, G.H. BAKKER and J.G.M. KLIJN, Endocrinology 12_.! 1375-1382 (1987). I. ROTHCHILD, Rec Progr Horm Res 37 183-283 (1981). J.Th.J. UILENBROEK, P.J.A. WOUTERSEN and P. VAN DER VAART, J Endocr 12_.._0325-330 (1989). W.G. WIEST, W.R. KIDWELL and K. BALOGH, Endocrinology 82 844-859 (1968). I. HASHIMOTO and W.G. WIEST, Endocrinology 84 886-892 (1969). S.A. LAMPRECHT, H.R. LINDNER and J.E. STRAUSS, Biochem Biophys Acta 187 133-143 (1969). M. LAHAV, S.A. LAMPRECHT, A. AMSTERDAM and H.R. LINDNER, Mol Cell Endocr 6 293302 (1977). M. DIMATTINA, B. ALBERTSON, D.E. SEYLER, D.L. LORIAUX and RJ. FALK, Contraception 34 199-206 (1986). M. DIMATTINA, B. ALBERTSON, V. TYSON, D.L. LORIAUX and R.J. FALK, Fertility and Sterility 48 229-233 (1987). F.J. ROJAS, J.L. O'CONNER and R.H. ASCH, J Ster Bioch 23 1053-1058 (1985). J.Th.J. UILENBROEK, J. Endocr. 12_._.29423-429 (1991). P. VAN DER SCHOOT, J.Th.J. UILENBROEK and E.J. SLAPPENDEL, J Reprod Fertil 87 593-601 (1989).
50, pp.
1173-1178
Pergamon
Press
INHIBITION OF INDUCTION OF 20g-HYDROXYSTEROID DEHYDROGENASE ACTIVITY IN RAT CORPORA LUTEA IN VITRO BY THE PROGESTERONE ANTAGONIST RU486 J.Th.J. Uilenbroek, P.J.A. Woutersen and B. Karels Department of Endocrinology and Reproduction, Faculty of Medicine, Erasmus University Rotterdam The Netherlands (Received
in final
form February
I0,
1992)
Summary To determine if the antiprogestagen RU486 has a direct effect on luteal progesterone secretion, whole corpora lutea or dispersed luteal cells were incubated in the presence of RU486. Whole corpora lutea, isolated from rats at day 5 of pseudopregnancy, were incubated individually in hormone-free medium. The concentrations of progesterone and 20n-dihydroprogesterone in the medium plus corpus luteum was measured before and after 24 h of incubation. In the absence of RU486 the concentration of 20~-dihydro-progesterone increased, while that of progesterone remained unchanged. In the presence of RU486 (230 I.tM) the concentration of both progesterone and 20a-dihydro-progesterone was increased. Dispersed luteal cells were incubated for 24 h in the presence of various amounts of RU486. In the absence and in the presence of 0.2 and 2.3 I.tM RU486 a high ratio between 20~dihydro-progesterone and progesterone was found, while in the presence of 23 ~M RU486 the concentrations of progesterone and 20c~-dihydro-progesterone were equal. 20~-Hydroxysteroid dehydrogenase (20~-HSD) activity measured in luteal homogenates started to increase between 6 and 12 h of incubation. This increase could be prevented after incubation of the corpora lutea in the presence of 23 or 230 I.tM RU486 for 24 hrs. It is concluded that the progesterone antagonist RU486 can have a direct effect on luteal progesterone production. RU486 prevents the increase in 20g-HSD activity that normally occurs during in vitro incubation. However, since these effects in vitro can only be obtained with high concentrations of RU486, it is unlikely that this antiluteolytic effect plays a role after injection of RU486 in vivo.
RU486 (•7B-hydr•xy-••B-[4-dimethy•-amin•pheny•]-•7a•[•-pr•pyny•]•estra-4•9-diene-3-•ne) is a synthetic 19-norsteroid that binds to the progesterone receptor with high affinity and acts as a progesterone antagonist (1). In humans and non-human primates it induces early menstruation when given during the luteal phase of the menstrual cycle (2,3) and abortion when given during early pregnancy (4,5). This effect is due to inhibition of receptor-mediated progesterone action on the uterus. However, progesterone receptors are also present in the ovary (6) and RU486 binds to this receptor with high affinity (7). Luteolytic and antiluteolytic effects of RU486 have been described (8,9). Most of these effects could be attributed to changes in circulating levels of prolactin. However, it has been hypothesized that progesterone also plays a role in the regulation of its own production (10). Therefore it is the objective of the present study to investigate whether RU486 might have a direct effect on luteal progesterone production. A possible direct effect of RU486 on the corpus luteum in vivo is difficult to assess as progesterone influences the secretion of gonadotrophins and prostaglandins, which are required for normal luteal function (10). Therefore the effect of RU486 on luteal activity has been studied in vitro. Correspondence should be addressed to Dr. J. Uilenbroek
Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press plc All rights reserved.
1174
RU486 and Corpus Luteum Activity
Vol.
50, No. 16, 1992
Materials and Methods Animals Adult Wistar rats (3-5 months old) were used in this study. The animals were housed under standard conditions of light (on: 05.00-19.00 h) and temperature (20-23°C). The females showed almost exclusively 5-day reproductive cycles as detected by vaginal smears, which were made 5 days a week. The animals were made pseudopregnant by caging them overnight with a vasectomized male. At day 5 of pseudopregnancy (the day of estrus was considered as the first day of pseudopregnancy) the animals were sacrificed, the ovaries were dissected out and the last generation of corpora lutea was isolated under a dissection microscope. The corpora lutea were either incubated intact or collected in Dulbecco's phosphate buffered saline (PBS) for the preparation of dispersed luteal cells. Incubation of whole corpora lutea Whole corpora lutea were incubated individually in 1 ml Medium-199 (Gibco, Grand Island, NY, USA), pH 7.4, containing Hepes (25 mM) (Sigma Chemical Co., St. Louis, MO, USA), antibiotic-antimycotic mixture (250 p.g amphotericin, 105 IU penicillin, 100 mg streptomycin sulfate per 1) (Gibco, Grand Island, NY, USA) and various amounts of RU486 (RousseI-Uclaf, France) dissolved in 10 ~1 ethanol. The incubations were carried out at 37°C under an atmosphere of 95% 02 and 5% CO 2 in a shaking waterbath. At the end of the incubation 1 ml ethanol was added to the medium and the corpus luteum was crushed. The mixture was stored at -20°C until measurement of the progesterone and 20a-dihydro-progesterone content. Dispersion of luteal cells Isolated corpora lutea from 3 to 4 rats (12-14 corpora lutea per rat) were washed in Dulbecco's PBS, pooled and incubated in 5 ml Medium Essential Medium (MEM, Gibco, Grand Island, NY, USA) containing 0 . 1 % collagenase, 0.02 % DNAse and 1 % fetal calf serum (FCS). Collagenase, DNAse and FCS were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The corpora lutea were incubated for two consecutive 30 min periods in a shaking waterbath at 37°C. After each incubation the corpora lutea were mechanically dispersed with a plastic Pasteur pipet. After the last incubation, tissue fragments were removed by filtration through cheesecloth. The dispersed cells wcrc collected by centrifugation (5 min, 800 g) and the pellet was washed three times with MEM containing 1% FCS. The pellet was resuspended in MEM containing 1% FCS in a final concentration of 1 corpus luteum per 100 ILl. The dispersed cells equivalent to 1 corpus luteum were incubated in 1 ml MEM containing 1% FCS and increasing amounts of RU486 (0.23 - 23 nmol) at 37°C in an humidified atmosphere of 5% CO 2 and 95% air. After incubation for 24 h the incubation medium was centrifuged (10 min, 2500g) and the supernatant was stored at -20°C for assay of progesterone and 20ndihydro-progesterone. Steroid assays The incubation medium containing the crushed corpora lutea was centrifuged for 10 min at 2500 g and the pellet was extracted twice with 500 l.tl acetone. The combined supernatants were evaporated to dryness and redissolved in 1 ml PBS (0.01 M, pH 7.0) containing 0.15% (w/v) gelatin. Progesterone and 20e-dihydroprogesterone concentrations were measured by radio-immunoassay as described previously (11). Since high amounts of RU486 were used in the incubation medium special attention was given to the validation of steroid measurements. Crossreactivity of RU486 with the progesterone and 20~-dihydroprogesterone antibodies was less than 0.01%. In a number of incubation samples containing RU486 (230 ~M), progesterone was measured with and without chomatography. The samples were extracted twice with hexaneaether (4:1, v/v). Thin layer chromatography (TLC) was performed using precoated silica-plates G F ~ (Merck, Darmstadt, Germany) as stationary phase and chloroform-acetone (95:5, v/v) as mobile phase. As samples measured with TLC were not lower than those measured without TLC, progesterone and 20n-dihydroprogesterone measurements in the presence of RU486 (230 p.M) were considered to be reliable. Enzyme assays For the measurement of 20ct-hydroxysteroid dehydrogenase (20ct-HSD) activity corpora lutea were weighed, homogenized in 0.05 M Trls/EDTA-buffered saline pH 7.4 and centrifuged at 800 g (5 min 4°C). One hundred I-d supcrnatant of a 3% homogenate was incubated with (4 x 104 d.p.m) [1,2,6,7,16,17 3H] progesterone (s.a. 140 C/mmol), 16 nmol progesterone, 500 I.tl of a NADPH-regenerating system (containing 1.4 U glucose 6-phosphate dehydrogenase, 20 mM glucose 6-phosphate and 2raM NADP) and 250 p.l of an inhibitor of aromatase activity (androsta-l.4.6-tricne-3.17-dione, ATD, 400 p.M) and an inhibitor of 5ctreductase (4-androsten-3-onc-17B carboxylic acid, AC, 398 p.M) in a final concentration of 1 ml Tris/EDTA.
Vol.
50, NO.
16, 1992
RU486 and Corpus Luteum Activity
1175
After incubation for 30 min in a shaking waterbath at 37°C the reaction was stopped by adding 1.5 ml ice-cold hexane-aether (4:1 v/v). After extraction twice with hexane-aether progesterone and 20=-dihydro-progesterone were separated by TLC as described above. The enzyme activity was expressed in pmol substrate converted per min per mg luteal tissue. 3H-labeled and unlabeled steroids used were obtained from Amersham International PLC (Amersham, UK). To investigate whether RU486 had a direct antagonistic effect on the 20n-HSD activity the enzyme activity was also measured in the presence of RU486. Corpora lutea were first incubated for 24 hrs in the absence of RU486. Thereafter a 3% luteal homogenate was incubated with increasing amounts of 3H-labeled progesterone and unlabeled progesterone as substrate in the presence and absence of 12 nmol RU486. Statistics Results are given as means ± S.E.M. Statistical analysis consisted of Student's t-test and one-way analysis of variance (ANOVA). If the overall test was significant, comparison between groups was made by the Least Significant Difference (LSD) test. A difference was considered significant if the probability (P) was <0.05 (two-tailed). Results Effect of RU486 on whole corpora lutea and dispersed luteal cells in vitro At day 5 of pseudopregnancy corpora lutea contained 0.27 ± 0.02 nmol progesterone and 0.13 ± 0.03 nmol 20ct-dihydro-progesterone per corpus luteum. After 24 h of incubation the concentration of progesterone in the corpus luteum plus medium remained constant (0.26 ± 0.02 nmol), while that of 20ct-dihydroprogesterone increased to 1.04 ± 0.1 nmol (P<0.01, Student's t-test). Incubation in the presence of RU486 (230 I.tM) resulted in high concentrations of both progesterone and 20et-dihydro-progesterone (1.30 ± 0.17 nmol and 1.45 ± 0.16 nmol respectively). Incubation of dispersed luteal cells in the absence and presence of 0.2 and 2.3 I~M RU486 for 24 h resulted in a low concentration of progesterone and a high concentration of 20ct-dihydro-progesterone in the medium (Fig. 1). However, incubation in the presence of 23 I.tM RU486 resulted in a significantly higher concentration of progesterone and a lower concentration of 20et-dihydro-progesterone (ANOVA; LSD=7.79 pmol for progesterone and 47.7 pmol for 20ct-dihydro-progesterone).
300
i
\\
200
\\\
:
\\\ \\\
\\
---e---
\
prog 20a-OHprog
100 o
0 0.1
1
10
100
RU48S ( . M )
Fig. 1. Concentrations of extracellular levels of progesterone (e) and 20¢t-dihydro-progesterone (o) after incubation of dispersed luteal cells for 24 h in the presence of increasing amounts of RU486. The data represent the mean ± S.E.M. of 4 incubations. When error bars are absent, they are too small to indicate.
1176
RU486 and Corpus Luteum Activity
Vol. 50, No. 16, 1992
Effect of RU486 on 20a-HSD activity The activity of 20a-HSD in homogenized corpora lutea was measured before and after 3, 6, 12, 24 and 48 h of incubation. Before incubation the activity was low (< 3 pmol/min/mg luteal tissue). Between 6 and 12 h of incubation the activity increased significantly (ANOVA; LSD=5.34 pmol) and was high after 24 and 48 h of incubation (Fig. 2). Incubation in the presence of RU486 for 24 h prevented the increase of 20a-HSD activity in a dose dependent manner (Table I). A concentrations of 2.3 I~M was already effective (ANOVA; LSD =3.50 pmol).
100 •
E
80
T
m
E 0
E
60
40 .I-
6
0
20
04
0 0
i
,
,
i
12
24
36
48
hrs of Incubation
Fig. 2. 20a-hydroxysteroid dehydrogenase (20a-HSD) activity in corpora lutea before and after incubation. The activity is measured in a 3% homogenate and is expressed as pmol/min/mg luteal tissue. The data represent the mean ± S.E.M. of triplicate measurements. When error bars are absent, they are too small to indicate.
TABLE 1 20a-Hydroxysteroid dehydrogenase (20a-HSD) activity in whole corpora lutea after 24 h of incubation in the absence and presence of RU486. The values represent the mean ± S.D. of duplicate measurements. RU486 (p.M)
-2.3 23 230
20a-HSD activity (pmol/min/mg)
46.4 ± 0.9 35.9 ± 1.6 14.6 ± 0.0 7.6 ± 0.6
Vol. 50, No. 16, 1992
RU486 and Corpus Luteum Activity
1177
To investigate whether the low 20a-HSD activity found after incubation with RU486 might be due to an inhibition of the induction of this enzyme or to a direct competitive inhibition of the conversion of progesterone to 20ct-dihydro-progesterone the following experiment was performed. Corpora lutea were first incubated for 24 h to ensure a high 20rt-HSD activity. Thereafter 20tz-HSD activity was measured in the presence and absence of RU486 (12 I~M). As the conversion rate between progesterone to 20--OH progesterone was not different between the two groups, it was concluded that the low 20~-HSD activity after 24 h of incubation in the presence of RU486 was not due to competitive inhibition of RU486 with the snbstrate progesterone on the enzyme. Discussion Corpora lutea of pseudopregnant rats secrete large amounts of progesterone. During luteolysis at the end of pseudopregnancy progesterone in the circulation decreases. This decrease is partially due to a decrease in progesterone production, but mainly to the conversion of progesterone to the biologically inactive metabolite 20n-dihydro-progesterone by the microsomal enzyme 20~t-HSD (12). The present study shows that the activity of this enzyme in corpora lutea increases during incubation in hormone-free medium, resulting in a low concentration of progesterone and a high concentration of 20adihydro-progesterone at the end of the incubation period. The increase starts after 6 h suggesting "de novo" synthesis. The mechanism by which 20¢t-HSD activity increases during incubation is not know. In vivo the activity is low in the presence of high prolactin levels (13) and increases as prolactin declines (14). From these data it is conceivable that the increase in vitro is due to the absence of prolactin in the culture medium. However, addition of prolactin to the culture medium did not prevent the increase in 20a-HSD activity (Uilenbroek, unpublished results), although such an effect has been reported by others (15). The present data show that the increase in 20ct-HSD activity is prevented by addition of RU486 to the culture medium. The low enzyme activity at the end of the incubation period with RU486 is not due to a competitive inhibition of the compound with progesterone on the enzyme. Rather the antiprogestagen inhibits the synthesis of this enzyme. From the inhibitory action of RU486 on the increase of 20tz-HSD activity it can be concluded that the increase in 20a-HSD activity in vitro is a progesterone-mediated event. An effect of progesterone on its own activity has been proposed by Rothchild (10). However, this author concluded that progesterone stimulates and does not inhibit its own production. The effect of RU486 on 20a-HSD activity can only be obtained with high concentrations of RU486. Large amounts might be required to block a possible autocrine effect of progesterone on its own metabolism. Furthermore, these high levels might be of relevance for in studies performed in vivo, adult rats were given daily injections with 2 mg/day. Assuming an equal distribution and no rapid metabolism corpora lutea in vivo are then exposed to I~M concentrations of RU486. An inhibition of 20~t-HSD activity by RU486 should result in an increased concentration of progesterone and a decreased concentration of 20ct-dihydro-progesterone. This was found in the medium after incubation of luteal cells with 23 o.M RU486. Incubation with whole corpora lutea however, resulted in high concentrations of both progesterone and 20a-dihydro-progesterone, while an increase in 20a-HSD activity was already prevented at 2.3 I~M RU486. This discrepancy between enzyme activity and the content of progesterone and 20tz-dihydro-progesterone in the whole corpora lutea might be related to the lower penetration rate of RU486 in the whole corpora lutea compared with dispersed cells. It is likely that in whole copora lutea a considerable amount of 20ct-dihydro-progesterone has already been formed before a further increase of 20ct-HSD activity occurs. A direct effect of RU486 on human ovarian steroidogenesis has been reported by Dimattina et al. (16,17). They found an inhibition of progesterone production by human granulosa cells due to inhibition of 3BHSD activity (16). Furthermore, a decrease in 17-hydroxylase activity with doses RU486 exceding 50 ~tM has also been reported (17). A direct effect of RU486 on LH receptor binding or cyclic AMP generation using a membrane preparation from human corpora lutea has not been found (18). A direct antiluteolytic effect of RU486 in vitro is in line with some results observed in vivo. Daily injections of RU486 (2 mg/rat/day) for 28 days result in an increase of serum progesterone levels (9). These high levels of progesterone however, are due to the high circulating levels of prolactin (19). Moreover, in lactating rats RU486 did not increase luteal progesterone production (20). In lactacting rats prolactin levels are high due to the suckling stimulus and are not different between RU486 treated rats and controls. So in vivo studies do not support a direct effect of RU486 on luteal progesterone production.
1178
RU486
and C o r p u s L u t e u m A c t i v i t y
Vol.
50, No.
16, 1992
In conclusion, incubation of corpora lutea with large amounts of RU486 results in an inhibition of the conversion of progesterone to 20~x-OH progesterone suggesting that progesterone itself might play a role in the increase of 20n-HSD activity. However, I.tM concentrations are required to obtain these effects. It is therefore unlikely that the effects found in vitro play a major role in the effects observed after injections of RU486 in vivo. Acknowledgement The authors are indebted to Dr. R. Deraedt (Roussel-Uclaf, France) for supplying RU486. References
2,
3. 4.
9.
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
D. PHILIBERT, M. MOGUILEWSKY, I. MARY, D. LECAQUE, C. TOURNEMINE, J. SECCHI and R. DERAEDT, The antiprogestin steroid RU486 and human fertility control, Baulieu E.E. and Segal S. (eds), 49-68, Plenum Press, New York, (1985). D.L. HEALY, E.E. BAULIEU and G.D. HOGDEN, Fertility and Sterility 40 253-257 (1983). B. SHORTLE, I. DYRENFURTH and M. FERIN, J Clin Endocrinol Metab 60 731-735 (1985). L. KOVACS, M. SAS, B.A. RESCH, G. UGOSCA1, M.L. SWAHN, M. BYGDEMAN and P.J. ROWE, Contraception 29 399-410 (1984). B. COUZINET, N. LE STRAT, A. ULMANN, E.E. BAULIEU and G. SCHAISON, New England Journal of Medicine 3!5 1565-1570 (1986). J.R. SCHREIBER and A.J.W. HSUEH, Endocrinology 10__._55915-919 (1979). J.R. SCHREIBER, AJ.W. HSUEH and E.E. BAULIEU, Contraception 28 77-85 (1983). T. KAWANO, H. OKAMURA, C. TAJIMA, K. FUKUMA and H. KATABUCHI, J Reprod Fert 83 279-285 (1988). P. VAN DER SCHOOT, G.H. BAKKER and J.G.M. KLIJN, Endocrinology 12_.! 1375-1382 (1987). I. ROTHCHILD, Rec Progr Horm Res 37 183-283 (1981). J.Th.J. UILENBROEK, P.J.A. WOUTERSEN and P. VAN DER VAART, J Endocr 12_.._0325-330 (1989). W.G. WIEST, W.R. KIDWELL and K. BALOGH, Endocrinology 82 844-859 (1968). I. HASHIMOTO and W.G. WIEST, Endocrinology 84 886-892 (1969). S.A. LAMPRECHT, H.R. LINDNER and J.E. STRAUSS, Biochem Biophys Acta 187 133-143 (1969). M. LAHAV, S.A. LAMPRECHT, A. AMSTERDAM and H.R. LINDNER, Mol Cell Endocr 6 293302 (1977). M. DIMATTINA, B. ALBERTSON, D.E. SEYLER, D.L. LORIAUX and RJ. FALK, Contraception 34 199-206 (1986). M. DIMATTINA, B. ALBERTSON, V. TYSON, D.L. LORIAUX and R.J. FALK, Fertility and Sterility 48 229-233 (1987). F.J. ROJAS, J.L. O'CONNER and R.H. ASCH, J Ster Bioch 23 1053-1058 (1985). J.Th.J. UILENBROEK, J. Endocr. 12_._.29423-429 (1991). P. VAN DER SCHOOT, J.Th.J. UILENBROEK and E.J. SLAPPENDEL, J Reprod Fertil 87 593-601 (1989).