Placenta (1991), 12,51 I-520
Evidence for Modulation of Progesterone Secretion by Calcium and Protein Kinase C Activators in Ovine Chorionic Cells M. P. DE LA LLOSA-HERMIER”, J. MARTAL’: A. RICOUR” & C. HERMIER” a C.2v.R.S., Laboratoire de Biochimie des Hormones, 91198 GifSW- Yvette cPdex,France b I.N.R.A., Unite’d’Endocrinologie de l*Embryon-Station de Ph.ysiologie animale, 78352Joq-en-J’osas cidex, France Paper accepted 164.1991
SUMMARY The hypothesis that calcium-dependent mechanisms may be i?tvolved in regulating ovine placental steroidogenesis was investigated using chorionic cells isolated by enzymatic digestion. Treatment ofthe cells with the calmodulin antagonist tri$uopermine (TFP) or pimozide caused a dose-related inhibition of progesterone (P4) production by 8Oper cent (P < 0.001) at 40~ TFPand 56per cent (P < 0.001) at IO,uMpimozide. Moreover, the conversion of25 hydroxycholesterol(25 OH Chol.) to P4 was impaired in the presence of these compounds. These experiments suggest the involvement of a calcium-calmodulin system in the regulation of ovine placental P4 synthesis. Interestingly, calcium ionophore A23187 caused a gradual decline in P4 secretion und completely blocked it at I w (P < 0.001) and remains absent even in the presence of 2.5 OH Chol. In contrast, EGTA increased P4 secretion (P < 0.01). Further, in the presence of 3 mM EGTA the inhibitory eflect of 1 pM A23187 was jtil[y reversed. Ta k en together these results suggest that extracellular calcium could play a role of negative modulation ofP4 secretion in these cells. The possible involvement of protein kinase C (PKC) was tested using tumorpromoting phorbol ester (PMA) orpermeant diacylglycerols (OAG or DOG). These compounds were unable to modiJjl basal P4 secretion but reduced 25 OH Chol stimulated secretion to basal level. Thephorbol ester that was unable to activate PKC had no efect on the metabolism of25 OH chol. Thus, P&L4 and diacylglycerol effects ure probably mediated by PKC. These data support the hypothesis that PKC activation plays a role in the modulation of cholesterol side-chain cleavage activity in ovine chorionic cells. These results show that calcium-dependent processes are involved in both positive and negative control ofP4 secretion by ovine placenta. Our results also suggest a role jar calmodulin and PKCpathwoys in modulating this secretion. 0143-4004/91/050511
+ 10 $05.00/O
0
1991 Baillike
Tindall Ltd
512
Placenta (1991), Vol. 12
INTRODUCTION Progesterone (P4) is considered to be essential to conceptus and foetal development throughout pregnancy but only in some species, such as human and sheep, is placenta able to secrete P4 in very early gestation. Although endocrine regulation of luteal P4 is relatively well known, the mechanisms controlling placental P4 secretion have not been fully elucidated. However, it has been shown that in human trophoblast, this process is regulated by calcium and protein kinase C (PKC) mediated signal-transduction pathways (Kasugai et al, 1987; Ritvos et al, 1988). In sheep, the placenta is the main source of P4 after day 50 of pregnancy. Data demonstrating significant changes in placental P4 secretion through pregnancy (Basset et al, 1969) have been interpreted as evidence of the existence of a mechanism controlling the secretion of this steroid (Thorbum, Challis and Robinson, 1977). Nevertheless, the physiological factors and intracellular signals responsible for modulation have not yet been identified. We recently reported (De La Llosa-Hermier et al, 1988) that luteinizing hormone, gonadotropin-releasing hormone or their putative second messengers failed to stimulate P4 synthesis during the first 3 h of incubation of placental explants. However, it is conceivable that this failure may be related to the time during which the tissue was exposed to the supposed regulators. Using longer experiments (6 h) and enzymatic dispersed cells, we have now re-examined the second messenger systems which appear to regulate the endocrine activity of other steroidogenic tissues (Nishizuka et al, 1984; Dufau, 1988) and investigated their possible involvement in the modulation of P4 secretion by ovine placenta. The involvement of calcium-dependent mechanisms was studied by monitoring the effects of several compounds: calmodulin antagonists (pimozide and trifluoperazine), calcium ionophore A23187 and extracellular calcium chelator EGTA. The role of protein kinase C (PKC) was also evaluated using factors known to activate this enzyme such as tumorpromoting phorbol esters (Castagna et al, 1982) and synthetic diacylglycerols (Kraft and Anderson, 1983). The time during gestation when the placentomes were obtained might be an important variable in establishing the mechanisms involved in the regulation of steroidogenic function. It has been reported that in sheep, placental secretion of P4 rises rapidly between days 90 and 120 of gestation (Thorbum, Challis and Robinson, 1977). This data, coupled with the observations that cotyledonary mass (and probably the number and proportion of giant and mononucleated cells of the chorion) remains practically stable at this period (Martal and Djiane, 1977; Wooding, 1982) suggests that the rate of synthesis of P4 may increase at this time. Thus, it might be expected that placenta after day 90 offers optimal conditions to examine factors that may act as second messengers in the regulation of P,+ secretion. Consequently the present study was carried out in foetal cotyledons between days 100 and 120 of pregnancy.
MATERIALS
AND
METHODS
Chemical products Calcium- and magnesium-free Hank’s balanced salt solution (HBSS) and medium 199 containing 25 mu Hepes (M199) were obtained from Gibco BRL (France). Trypsin was purchased from ICN Biochemicals (Cleveland, OH). Hyaluronidase type II (375 units/mg), DNase I (2100 units/mg), Soybean trypsin inhibitor type 11s (lo4 BAEE units/mg),
De La Lhsu-Hmier
et al: Modulation ofprogesterone Secretion by Cal&m
513
ionophore A23 187, phorbol 12-myristate- 13-acetate (PMA), phorbol 13 monoacetate (PM), 1,2-dioctanoyl-sn-glycerol (DOG), 1-oleoyl-2 acetyl-sn-glycerol (OAG) and ethylene glycol-bis @-aminoethyl ether)-N,N,N,N’-tetraacetic acid (EGTA) were supplied by Sigma Chemical Co. (St Louis, MO). Trifluoperazine (TFP) was a generous gift of Theraplix Laboratory (Gien, France) and pimozide was supplied by Janssen Laboratories (Paris, France). S-cholesten-3B, 25-diol (25 OH Chol) was purchased from Steraloids (Wilton, NH.). Cell dispersion Pregnant ewes of Prealpes du Sud breed were killed between day 100 and day 120 of pregnancy. Foetal cotyledons were separated manually from maternal cotyledons, the haemophogous zone was removed and the tissue washed several times with cold 0.154 .\f NaCl to remove any remaining blood. The tissue fragments were first digested in 4-S volumes of HBSS medium containing 0.2 per cent trypsin and 0.1 per cent hyaluronidase for 10 min at 36°C. The supernatant was discarded and the remaining tissue was subjected to two successive 20-25 min digestion periods with fresh HBSS containing 0.1 per cent trypsin and 0.02 per cent DNase. The resultant cell suspensions were filtered through two layers of cheese cloth, the cells collected by centrifugation (300 g, 5 min, at room temperature) washed once in HBSS containing soybean trypsin inhibitor (0.5 mg/ml) washed again in Ml99 and resuspended in this medium. Cell numbers were estimated with a hemocytometer and just before the diluted to l-2 x lo6 cells/ml. Cell viability, estimated microscopically experiment by trypan blue dye exclusion ranged from 80 to 90 per cent. It was not affected by 6 h-incubation. Incubation Samples of dispersed chorionic cells (0.4-0.6 X 1O6cells) were incubated in a final volume of 0.5 ml hII for 6 h at 37°C in a humid atmosphere of 5 per cent CO*/95 per cent air. At the end of the incubation period, the samples (medium and cells combined) were quickly frozen and stored at -20°C until assayed for P4 (6 h incubated samples). In order to determine the initial P+ content in the cells, samples were frozen at zero time (non-incubated samples). A23187 and 25 OH Chol were dissolved in ethanol and the phorbol esters or synthetic diacylglycerols were dissolved in dimethylsulphoxide (DMSO). Each sample received the same concentration of ethanol and DMSO. The final concentration of ethanol was 1 per cent and that of DMSO did not exceed 0.1 per cent. Neither had a measurable effect on P.+ production at these concentrations. Progesterone assay P4 concentrations were determined by radioimmunoassay (RIA) as previously described (De La Llosa-Hermier et al, 1988). Before RIA, non-incubated and incubated samples were thawed, sonicated for 15 set, and centrifuged (2000 g, 15 min, 4°C) to remove particulate matter. The supernatant obtained served for steroid measurements. The P4 antiserum was purchased from the Institute Pasteur (Paris). The mean association constant of antiserum for P4 was 1.3 x lo-” M-I; non-specific binding was less than 4 per cent; assay sensitivity was 0.1 ng/ml; the intraassay coefficient of variation was less than 5 per cent. Statistical analysis The results represent means * s.d. of triplicate samples from a representative experiment. For each experiment, similar results were obtained in at least two or three experiments. Data
Placenta (1991), Vol. 12
514
were first analysed by the analysis of variance (ANOVA). When significant differences were observed (I’< 0.05) Student’s t-test was used to compare the control group to treatment groups.
RESULTS Effects of calcium
on P4 synthesis
We first examined the effects of increasing concentrations of two potent calmodulin inhibitors, pimozide and TFP. The results presented in Figure l(a) show that both compounds induced a concentration-dependent inhibition of basal P4 secretion. TFP (40 pM) inhibited P4 production by 80 per cent (P < 0.001) and pimozide (10 ,UM) by 56 per cent (P < 0.01). To determine if these compounds affected P450 see activity (cholesterol side chain cleavage enzyme system), 2.5 OH Chol was used. This hydroxy-steroid, which bypasses the step that regulates the movement of cholesterol to the site of P450 see catalysed scission, is an effective substrate for.this enzymatic system (Taoff, Schleyer and Strauss, 1982). Since 38 hydroxysteroid dehydrogenase/574 isomerase activity is not rate-limiting in 3.5 2 8
(a) 3.0 *
“0
2.5
p
2.0
Evidence for Modulation of Progesterone Secretion by Calcium and Protein Kinase C Activators in Ovine Chorionic Cells M. P. DE LA LLOSA-HERMIER”, J. MARTAL’: A. RICOUR” & C. HERMIER” a C.2v.R.S., Laboratoire de Biochimie des Hormones, 91198 GifSW- Yvette cPdex,France b I.N.R.A., Unite’d’Endocrinologie de l*Embryon-Station de Ph.ysiologie animale, 78352Joq-en-J’osas cidex, France Paper accepted 164.1991
SUMMARY The hypothesis that calcium-dependent mechanisms may be i?tvolved in regulating ovine placental steroidogenesis was investigated using chorionic cells isolated by enzymatic digestion. Treatment ofthe cells with the calmodulin antagonist tri$uopermine (TFP) or pimozide caused a dose-related inhibition of progesterone (P4) production by 8Oper cent (P < 0.001) at 40~ TFPand 56per cent (P < 0.001) at IO,uMpimozide. Moreover, the conversion of25 hydroxycholesterol(25 OH Chol.) to P4 was impaired in the presence of these compounds. These experiments suggest the involvement of a calcium-calmodulin system in the regulation of ovine placental P4 synthesis. Interestingly, calcium ionophore A23187 caused a gradual decline in P4 secretion und completely blocked it at I w (P < 0.001) and remains absent even in the presence of 2.5 OH Chol. In contrast, EGTA increased P4 secretion (P < 0.01). Further, in the presence of 3 mM EGTA the inhibitory eflect of 1 pM A23187 was jtil[y reversed. Ta k en together these results suggest that extracellular calcium could play a role of negative modulation ofP4 secretion in these cells. The possible involvement of protein kinase C (PKC) was tested using tumorpromoting phorbol ester (PMA) orpermeant diacylglycerols (OAG or DOG). These compounds were unable to modiJjl basal P4 secretion but reduced 25 OH Chol stimulated secretion to basal level. Thephorbol ester that was unable to activate PKC had no efect on the metabolism of25 OH chol. Thus, P&L4 and diacylglycerol effects ure probably mediated by PKC. These data support the hypothesis that PKC activation plays a role in the modulation of cholesterol side-chain cleavage activity in ovine chorionic cells. These results show that calcium-dependent processes are involved in both positive and negative control ofP4 secretion by ovine placenta. Our results also suggest a role jar calmodulin and PKCpathwoys in modulating this secretion. 0143-4004/91/050511
+ 10 $05.00/O
0
1991 Baillike
Tindall Ltd
512
Placenta (1991), Vol. 12
INTRODUCTION Progesterone (P4) is considered to be essential to conceptus and foetal development throughout pregnancy but only in some species, such as human and sheep, is placenta able to secrete P4 in very early gestation. Although endocrine regulation of luteal P4 is relatively well known, the mechanisms controlling placental P4 secretion have not been fully elucidated. However, it has been shown that in human trophoblast, this process is regulated by calcium and protein kinase C (PKC) mediated signal-transduction pathways (Kasugai et al, 1987; Ritvos et al, 1988). In sheep, the placenta is the main source of P4 after day 50 of pregnancy. Data demonstrating significant changes in placental P4 secretion through pregnancy (Basset et al, 1969) have been interpreted as evidence of the existence of a mechanism controlling the secretion of this steroid (Thorbum, Challis and Robinson, 1977). Nevertheless, the physiological factors and intracellular signals responsible for modulation have not yet been identified. We recently reported (De La Llosa-Hermier et al, 1988) that luteinizing hormone, gonadotropin-releasing hormone or their putative second messengers failed to stimulate P4 synthesis during the first 3 h of incubation of placental explants. However, it is conceivable that this failure may be related to the time during which the tissue was exposed to the supposed regulators. Using longer experiments (6 h) and enzymatic dispersed cells, we have now re-examined the second messenger systems which appear to regulate the endocrine activity of other steroidogenic tissues (Nishizuka et al, 1984; Dufau, 1988) and investigated their possible involvement in the modulation of P4 secretion by ovine placenta. The involvement of calcium-dependent mechanisms was studied by monitoring the effects of several compounds: calmodulin antagonists (pimozide and trifluoperazine), calcium ionophore A23187 and extracellular calcium chelator EGTA. The role of protein kinase C (PKC) was also evaluated using factors known to activate this enzyme such as tumorpromoting phorbol esters (Castagna et al, 1982) and synthetic diacylglycerols (Kraft and Anderson, 1983). The time during gestation when the placentomes were obtained might be an important variable in establishing the mechanisms involved in the regulation of steroidogenic function. It has been reported that in sheep, placental secretion of P4 rises rapidly between days 90 and 120 of gestation (Thorbum, Challis and Robinson, 1977). This data, coupled with the observations that cotyledonary mass (and probably the number and proportion of giant and mononucleated cells of the chorion) remains practically stable at this period (Martal and Djiane, 1977; Wooding, 1982) suggests that the rate of synthesis of P4 may increase at this time. Thus, it might be expected that placenta after day 90 offers optimal conditions to examine factors that may act as second messengers in the regulation of P,+ secretion. Consequently the present study was carried out in foetal cotyledons between days 100 and 120 of pregnancy.
MATERIALS
AND
METHODS
Chemical products Calcium- and magnesium-free Hank’s balanced salt solution (HBSS) and medium 199 containing 25 mu Hepes (M199) were obtained from Gibco BRL (France). Trypsin was purchased from ICN Biochemicals (Cleveland, OH). Hyaluronidase type II (375 units/mg), DNase I (2100 units/mg), Soybean trypsin inhibitor type 11s (lo4 BAEE units/mg),
De La Lhsu-Hmier
et al: Modulation ofprogesterone Secretion by Cal&m
513
ionophore A23 187, phorbol 12-myristate- 13-acetate (PMA), phorbol 13 monoacetate (PM), 1,2-dioctanoyl-sn-glycerol (DOG), 1-oleoyl-2 acetyl-sn-glycerol (OAG) and ethylene glycol-bis @-aminoethyl ether)-N,N,N,N’-tetraacetic acid (EGTA) were supplied by Sigma Chemical Co. (St Louis, MO). Trifluoperazine (TFP) was a generous gift of Theraplix Laboratory (Gien, France) and pimozide was supplied by Janssen Laboratories (Paris, France). S-cholesten-3B, 25-diol (25 OH Chol) was purchased from Steraloids (Wilton, NH.). Cell dispersion Pregnant ewes of Prealpes du Sud breed were killed between day 100 and day 120 of pregnancy. Foetal cotyledons were separated manually from maternal cotyledons, the haemophogous zone was removed and the tissue washed several times with cold 0.154 .\f NaCl to remove any remaining blood. The tissue fragments were first digested in 4-S volumes of HBSS medium containing 0.2 per cent trypsin and 0.1 per cent hyaluronidase for 10 min at 36°C. The supernatant was discarded and the remaining tissue was subjected to two successive 20-25 min digestion periods with fresh HBSS containing 0.1 per cent trypsin and 0.02 per cent DNase. The resultant cell suspensions were filtered through two layers of cheese cloth, the cells collected by centrifugation (300 g, 5 min, at room temperature) washed once in HBSS containing soybean trypsin inhibitor (0.5 mg/ml) washed again in Ml99 and resuspended in this medium. Cell numbers were estimated with a hemocytometer and just before the diluted to l-2 x lo6 cells/ml. Cell viability, estimated microscopically experiment by trypan blue dye exclusion ranged from 80 to 90 per cent. It was not affected by 6 h-incubation. Incubation Samples of dispersed chorionic cells (0.4-0.6 X 1O6cells) were incubated in a final volume of 0.5 ml hII for 6 h at 37°C in a humid atmosphere of 5 per cent CO*/95 per cent air. At the end of the incubation period, the samples (medium and cells combined) were quickly frozen and stored at -20°C until assayed for P4 (6 h incubated samples). In order to determine the initial P+ content in the cells, samples were frozen at zero time (non-incubated samples). A23187 and 25 OH Chol were dissolved in ethanol and the phorbol esters or synthetic diacylglycerols were dissolved in dimethylsulphoxide (DMSO). Each sample received the same concentration of ethanol and DMSO. The final concentration of ethanol was 1 per cent and that of DMSO did not exceed 0.1 per cent. Neither had a measurable effect on P.+ production at these concentrations. Progesterone assay P4 concentrations were determined by radioimmunoassay (RIA) as previously described (De La Llosa-Hermier et al, 1988). Before RIA, non-incubated and incubated samples were thawed, sonicated for 15 set, and centrifuged (2000 g, 15 min, 4°C) to remove particulate matter. The supernatant obtained served for steroid measurements. The P4 antiserum was purchased from the Institute Pasteur (Paris). The mean association constant of antiserum for P4 was 1.3 x lo-” M-I; non-specific binding was less than 4 per cent; assay sensitivity was 0.1 ng/ml; the intraassay coefficient of variation was less than 5 per cent. Statistical analysis The results represent means * s.d. of triplicate samples from a representative experiment. For each experiment, similar results were obtained in at least two or three experiments. Data
Placenta (1991), Vol. 12
514
were first analysed by the analysis of variance (ANOVA). When significant differences were observed (I’< 0.05) Student’s t-test was used to compare the control group to treatment groups.
RESULTS Effects of calcium
on P4 synthesis
We first examined the effects of increasing concentrations of two potent calmodulin inhibitors, pimozide and TFP. The results presented in Figure l(a) show that both compounds induced a concentration-dependent inhibition of basal P4 secretion. TFP (40 pM) inhibited P4 production by 80 per cent (P < 0.001) and pimozide (10 ,UM) by 56 per cent (P < 0.01). To determine if these compounds affected P450 see activity (cholesterol side chain cleavage enzyme system), 2.5 OH Chol was used. This hydroxy-steroid, which bypasses the step that regulates the movement of cholesterol to the site of P450 see catalysed scission, is an effective substrate for.this enzymatic system (Taoff, Schleyer and Strauss, 1982). Since 38 hydroxysteroid dehydrogenase/574 isomerase activity is not rate-limiting in 3.5 2 8
(a) 3.0 *
“0
2.5
p
2.0
