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Evidence for modulation of progesterone secretion by calcium and protein kinase C activators in ovine chorionic cells
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
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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),
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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
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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 *
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0.
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Nonincubated samples
Control
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TFP Ptmozlde L 75PM25 OH Chol :............................................,.......: 6 h Incubated
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Effect of calmodulin antagonist TFP or pimozide on P+ secretion by ovine chorionic cells. (a) Basal P4 secretion in the presence of increasing concentrations of TFP or pimozide. (b) 25 OH Chol-dependent P4 secretion in the presence of TFP or pimozide. The values given are means + s.d. of triplicate samples. *PC 0.05; **P < 0.01; ***P < 0.001 compared to control value.
Figure 1.
De La Llosa-Her&r
et al: Modulation of ProgesteroneSecretion by Calcium 3.5r
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c
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” Non- Control 2 rn~ 3 rn~ 4 rn~ Incubated L samples EGTA
samples
2mm I.
3mkl
4mu
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:....
I /LLMA 23187 . . . . . . . . . . . ..__...._.._.....................................~ 6 h Incubated
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Figure 2. (a) Effect of increasing concentrations of calcium ionophore A23187 on P+ secretion by ovine chorionic cells. (b) Effect of different concentrations of EGTA in the absence or in the presence of A23 187. The values given are means + s.d. of triplicate samples. **P < 0.01; ***P < 0.001 compared to control value.
ovine placenta (De La Llosa-Hermier et al, 1988) 25 OH Chol conversion to P4 provides information about maximal capacity of steroidogenesis at the level of P450 see complex. Addition of 25 OH Chol to the incubation medium enhanced, in a dose-dependent manner, the amount of P4 synthesised by ovine chorionic cells during a 6 h incubation, with maximal effect attained at about 2.5 HIM(data not shown). Figure 1 (b) shows that in the presence of 75 ,UM25 OH Chol, P+ secretion was significantly (P < 0.05) increased while in the presence of 25 OH Chol plus TFP (40,~M) or pimozide (~O,U,M),P4 production was similar to that observed in control cells. These results indicate that the inhibitory effect of calmodulin antagonists could be explained, at least in part, on the basis of an alteration of P4.50 see activity. We then explored the influence of calcium ionophore A23187 on P4 secretion [Figure 2(a)]. Interestingly, A23 187 induced a dose-dependent inhibition of P,+secretion, which was almost complete in the presence of 1 plw A23187 (about 80 per cent, P < 0.001). It was of interest to evaluate the possible role of extracellular calcium in the demonstrated effects of A23187. Chelation of extracellular calcium by 0.5 or 1 mu EGTA had no significant effect on P4 synthesis (data not shown). However, the presence of 2 mM EGTA caused an increase (about 1.8-fold, P < 0.01) in P4 production compared with that of
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3.5 r 3.0 2 t
2.5
*$
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Nonincubated samples
Control
50 /LM
5OP 25 OH Chol
LII
3 mM EGTA 6 h Incubated
5OcLy 25 OH Chol I PM A 23187
somples
3. Effects of EGTA and calcium ionophore A23 187 in 25 OH Chol-dependent P4 secretion. The values given are means f s.d. of triplicate samples. ‘P < 0.05; ***P < 0.001 compared to control value.
Figure
untreated cells, and a plateau was obtained between 2 and 4 mu EGTA [Figure 2(b)]. Further, concomitant presence of EGTA and A23 187 led to a reversal of the inhibitory effect of the ionophore, depending on the concentration of EGTA used. While inhibition (P < 0.001) of P4 secretion was observed in the presence of 1 PM A23187 alone, stimulation (P < 0.001) appeared in the presence of 1 PM A23 187 plus 4 mM EGTA. Taken together these results suggest that extracellular calcium could play a role of negative modulator of ovine placental progesterone synthesis. We also tested the effects of EGTA and A23 187 on the conversion of 25 OH Chol to P4 (Figure 3). In this experiment 3 mu EGTA and 50 PM 25 OH Chol sign&&y stimulated basal P4 production (stimulation by EGTA 0.9-fold P < 0.05, by 25 OH Chol 1.4-fold P -=c0.001 and by 25 OH Chol plus EGTA 2.5-fold P -=c0.001). In contrast, there was a significant (P < 0.001) decrease in P4 secretion in the presence of 1 PM A23187 and also in the presence of 1 ,uM A23 187 plus 50 ,uM 25 OH Chol. Effects of PKC activators on P4 synthesis Addition of synthetic diacylglycerols OAG and DOG at 1,lO or 100 ,L~Mdid not significantly affect basal P4 secretion (data not shown). Figure 4(a) shows that a lack of effect was also observed in the presence of PMA. However, 25 OH Chol stimulated P4 secretion (P < 0.01) was reduced to basal level. Similar effects were observed in the presence of 100~~ DOG, while the non-tumor promoting phorbol ester PM, was unable to affect this secretion [Figure 4(b)]. In addition, over the same concentration range PMA also decreased EGTA-enhanced P4 secretion which remained at a basal level in the presence of 160 no PMA (Figure 5).
DISCUSSION This study demonstrates that calcium plays a key role in the regulation of P4 secretion by ovine chorionic cells. Several strands of evidence presented here suggest that calcium-
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ES I .5 0 iI I .o : F
0.5 0
Non- Control 1.6 “M 16 no 160~ I mcubated samples PMA
d
1.6 nM 16 no 160~4
1
,
PMA
L---J 50 /LLM25 OH Chol :. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 h Incubated samples
0 Nonincubated sample
Control 100 FLM 200 no DOG
PM
100 FLM 200 “M DOG
PM
50 /.LLM25 OH Chol ~..._...__...............................,,,.............~ 6h
Incubated
samples
concentrations of PMA on basal and 25 OH Chol-dependent P4 secretion by ovine placental cells. (b) Effects ofDOG and PM on basal and 25 OH Chol-dependent P4 secretions. The values given are means t s.d. of triplicate samples. **Pi 0.01; ***P < 0.001 compared to control value.
&we
4. (a) Effect ofincreasing
dependent processes are involved in both the positive and the negative control of this secretion. First, TFP or pimozide, at concentrations similar to those which bind to calmodulin, inhibited basal P4 synthesis: it therefore seems likely that the observed inhibition results from their ability to interfere with calmodulin-dependent process. Involvement of calcium-calmodulin pathway in P4 secretion has also been demonstrated in human (Kasugai et al, 1987) and bovine (Ullmann and Reimers, 1989) placenta as well as in Leydig cells (Hall, Osawa and Mrotek, 198 1). Although the intracellular mechanism(s) underlaying the control of steroidogenesis by this pathway are not completely elucidated, it has been shown that in Leydig cells calcium-calmodulin enhanced the transport of cholesterol to the mitochondria and P450 see activity. To investigate the impact of calcium-calmodulin system on this enzymatic complex, we used 25 OH Chol. In previous work (De La Llosa-Hermier et al, 1988) we showed that addition of 25 OH Chol did not increase P4 secretion by ovine explants during the first 3 h of incubation. Our present observations of a 25 OH Chol-conversion to P4 by dispersed chorionic cells during 6 h experiments suggest that in these conditions not enough endogenous substrate is available and/or cannot be mobilized to bring about the highest rate of P4 secretion possible. Since P4 production was not significantly increased by
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0
Nanincubated samples
Control
1.6 nkl
1
16nkl
L----_-J
3 mM EGTA
:.. . . . . . . 6 h Incubated
160nu
I
PMA
.:
samples
of increasing concentrations of PMA on EGTA-stimulated P4 secretion by ovine chorionic cells. The values are means + s.d. of triplicate samples. *P < 0.05; *#P < 0.01; *++P < 0.001 compared to control value.
Figure 5. Effect
25 OH Chol when calmodulin antagonists were added concomitantly, it may be expected that the calcium-calmodulin system plays a role in regulating the content and/or the activity of P450 see enzyme. The additional possibility of treatment-induced changes in cholesterol availability remains to be investigated. Second, it would be expected from data presented here, that agents which increase intracellular calcium concentration such as ionophores, could stimulate P4 secretion by ovine chorionic cells. This was the case in Leydig cells (Lin, 1985) or granulosa cells (Wang and Leung, 1987). Surprisingly, in the present work A23187 was able to inhibit P4 production. Although this effect might be non-specific, this is unlikely, because chelation of extracellular calcium by EGTA prevented the effect of A23 187, suggesting that it was totally dependent on calcium influx from extracellular calcium. Further, EGTA stimulates progesterone secretion. Taken together these results may be interpreted on the basis of a critical pool of calcium readily exchangeable with extracelhtlar fluid, which imposes a limit on ovine placental steroidogenesis. A23187 has also been reported to inhibit basal P4 synthesis in ovine luteal cells (Conley and Ford, 1989) and large bovine luteal cells (Alila, Dowd and Corradino, 1988). However, in human (Kasugai et al, 2987) and in bovine (Ullmann and Reimers, 1989), placenta A23 187 increases P4 secretion. Hence, ionophore induces a wide variety of effects depending on the cells or species studied. From the experiments presented here, it is proposed that calcium-dependent processes may limit P4 secretion in ovine chorionic cells by decreasing P450 see activity and also by decreasing the availability of substrate. Since our results with 25 OH Chol demonstrate that P4 production is substrate limited, it may be concluded that in the presence of EGTA, substrate availability is increased. Support for action on P450 see activity was provided by the fact that the inhibitory effect of A23 187 cannot be overcome by addition of 25 OH Chol, while 25 OH Chol metabolism was enhanced by EGTA. Another hypothetical mechanism is proposed by Ghosh et al (1938) who demonstrated that during the. loss of steroidogenic function observed in hCG-
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desensitized rat luteal cells, availability of cholesterol substrate and P450 see amounts was unaffected. These authors suggested that a substance might inhibit P450 see enzyme. Whether such a substance is involved in calcium-mediated inhibition of P4 production by ovine chorionic cells, remains to be studied. As alluded to above, results obtained with ionophore and EGTA appear to conflict with those obtained in the presence of TFP or pimozide. However, this discrepancy might only be apparent. Of particular interest is the observation that, although most calmodulin-regulated enzymes bind calmodulin in the presence of calcium, calmodulin can bind phosphorylase kinase even in absence of calcium (Cohen et al, 1980). On the other hand the cell mixture used is not homogeneous and may contain steroidogenic cells which might differ in the mechanisms involved in the regulation of P4 secretion. Finally, we have shown that treatment by PKC activators, either PMA or OAG or DOG, failed to affect basal P4 synthesis. However, EGTA or 25 OH Chol failed to significantly increase basal P4 secretion in the presence of PKC activators. Thus, it appears that the highest rates of P4 production by these cells would be strong evidence of the negative effect exerted by these compounds. Since PMA and synthetic diacylglycerols are believed to affect cell functions by activating PKC, these results support the concept that activation of PKC decreases P4 production by ovine chorionic cells. An inhibitory effect of PMA on steroidogenesis has also been reported in swine granulosa (T;‘eldhuis and Demers, 1986) and ovine luteal cells (Jammes, De La Llosa-Hermier and Hermier, 1988; Wiltbank, Knickerbocker and Niswender, 1989; Conley and Ford, 1989). In agreement with these studies, our present data suggest that PKC inhibits P450 see activity in ovine chorionic cells. It is interesting to note that A23187 is able to. stimulate PKC activation (Nishizuka et al, 1984). Thus, the observed ionophore-induced decrease of P4 secretion by ovine chorionic cells might be due to activation of PKC. However, because A23 187 provokes a greater inhibition than PMA it may be concluded that, at least in part, the inhibition induced by A23 187 is independent of PKC activation. In conclusion, we have shown that ovine placenta is not autonomous in terms of P+ secretion. There is ample evidence to support the concept that calcium plays a crucial role in modulating steroidogenesis in this tissue. Furthermore calmodulin and protein kinase C may be important components of the biochemical mechanisms involved in P4 secretion. At this time we are unaware of hormones and factors which under physiological conditions control the calcium-signalling system.
ACKNOWLEDGEMENTS This study was financially supported (Fresnes, Paris).
in part by Grant 9189012 from ‘Fondation
de la Recherche
en Hormonologie’
REFERENCES Alila, H. W., Dowd, J. & Corradino, R. A. (1988) Role of calcium in progesterone synthesis by small and large bovine luteal cells. 21st Annual Meeting of the Society for the Study of Reproduction, Seattle WA, p. 55 (Abstract). Basset, J. M., Oxborrow, T. J., Smith, J. D. & Thorbum, G. D. (1969) The concentration ofprogresterone in the peripheral plasma of the pregnant ewe.Jofounzal ofEndocrikology, 45,449-457. Castagna, M., Takai, Y., Kaibuchi, K., Sane, K., Kikkawa, U. & Nishizuka, Y. (1982) Direct activation of calcium activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. Journal of Biological Chemistry, 257, 7847-785 1.
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Cohen, P., Klee, C. B., Picton, C. & Shenohkar, S. (1980) Calcium control of muscle phosphorylase kinase through the combined action of calmodulin and troponin. Annals of the New YorkAcademy of Sciences, 356, 1Sl161. Conley, A. J. & Ford, S. P. (1989) Effects of TPA, A23187 and prostaglandin Fan on progesterone synthesis by dispersed-luteal cells. BiologyofReproduction, 40, i224-1230. _ De La Llosa-Hermier. M. P.. Zonznzo. M. A.. Martal. 1. & Hermier. C. (1988) Lack of short-term modulation ’ of in vitro placental progesterone iecretion in sheep. P&nta, 9,623A3 1.’ Dufau, M. L. (1988) Endocrine regulation and communicating functions of the Leydig cell. Annual Review of Physiology,50,483-508.
Ghosh, D. K., Peegel, H., Dunham, W. R., Sands, R. H. & Menon, K. M. J. (1988) Modulation ofprogesterone synthesis and cytochrome P450 levels in rat luteal cells during human chorionic gonadotropin-induced desensitized state. Endorrinology,123,514522. Hall, P. F., Osawa, S. & Mrotek, J. (1981) The influence of calmodulm on steroid synthesis in Leydig cells from rat testis. Enducrinologv,109, 1677-1682. Jammes, H.. De La Llosa-Hermier, M. P. & Hermier, C. (1988) Contr6le negatif de la sdroidogenese du corps jaune ovine par la PMA. PathologieiBiologie,36, abstract 8. Kasuzzi. M.. Kato. H.. IrIvnma H.. Kato. M.. Ninatmwa. T. & Tomoda Y. (1987) The role of Ca2+ and adeiosine 3’,5 ‘-monophoiphate’in the regtdatidn of progesterone production’by human’placental tissue.joburnal of ClinicalEndocrinologyand Metabolism, 65, 122-126. Kraft, A. S. & Anderson, W. B. (1983) Phorbol esters increase the amount of calcium Ca2+ phospholipiddependent protein kinase associated with plasma membrane. Nature, 301,621-623. Lin, T. (1985) The role of calcium/phospholipid-dependent protein kinase in Leydig cell steroidogenesis. Endocrinology,117,119-126. Martal, J. & Djiane, J. (1977) The production of chorionic somatomammotrophm in sheep.3oumal OfReproduction and Fertility, 49,285-289.
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Wooding, F. B. P. (1982) The role of the binucleate cell in ruminant placental structure.~oumalofReprodu&n Fertility, 31,31-39.
and
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
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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),
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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
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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
<
*c
c*+
cc
c+
f
E
1.5
v
1.0
:
0.5 0
Nonlncubatlon samples
Control 0.4j~M ~,LLM 40~
0.
I ~CMI PM 10~~
TFP Plmozlde ~.............................._____......._....,_._._..,.._~ 6 h Incubated
samples
3.5 2 8 “0 q s” 0 6 k : E
3.0 c
2.5 2.0 1.5 1.0 0.5 0
Nonincubated samples
Control
40/‘M
101”
TFP Ptmozlde L 75PM25 OH Chol :............................................,.......: 6 h Incubated
samples
Effect of calmodulin antagonist TFP or pimozide on P+ secretion by ovine chorionic cells. (a) Basal P4 secretion in the presence of increasing concentrations of TFP or pimozide. (b) 25 OH Chol-dependent P4 secretion in the presence of TFP or pimozide. The values given are means + s.d. of triplicate samples. *PC 0.05; **P < 0.01; ***P < 0.001 compared to control value.
Figure 1.
De La Llosa-Her&r
et al: Modulation of ProgesteroneSecretion by Calcium 3.5r
z
3.0
$
2.5
E
2.0
: @ E F
515
c
(a)
1.5 1.0 0.5 0
Nonincubated samples
Control
O.OI/LM
I
O.l/.LM
i/w
I
A 23187 :................I......__......,..._...__._.......: 6 h Incubated
” Non- Control 2 rn~ 3 rn~ 4 rn~ Incubated L samples EGTA
samples
2mm I.
3mkl
4mu
1
EGTA L-_-L
:....
I /LLMA 23187 . . . . . . . . . . . ..__...._.._.....................................~ 6 h Incubated
samples
Figure 2. (a) Effect of increasing concentrations of calcium ionophore A23187 on P+ secretion by ovine chorionic cells. (b) Effect of different concentrations of EGTA in the absence or in the presence of A23 187. The values given are means + s.d. of triplicate samples. **P < 0.01; ***P < 0.001 compared to control value.
ovine placenta (De La Llosa-Hermier et al, 1988) 25 OH Chol conversion to P4 provides information about maximal capacity of steroidogenesis at the level of P450 see complex. Addition of 25 OH Chol to the incubation medium enhanced, in a dose-dependent manner, the amount of P4 synthesised by ovine chorionic cells during a 6 h incubation, with maximal effect attained at about 2.5 HIM(data not shown). Figure 1 (b) shows that in the presence of 75 ,UM25 OH Chol, P+ secretion was significantly (P < 0.05) increased while in the presence of 25 OH Chol plus TFP (40,~M) or pimozide (~O,U,M),P4 production was similar to that observed in control cells. These results indicate that the inhibitory effect of calmodulin antagonists could be explained, at least in part, on the basis of an alteration of P4.50 see activity. We then explored the influence of calcium ionophore A23187 on P4 secretion [Figure 2(a)]. Interestingly, A23 187 induced a dose-dependent inhibition of P,+secretion, which was almost complete in the presence of 1 plw A23187 (about 80 per cent, P < 0.001). It was of interest to evaluate the possible role of extracellular calcium in the demonstrated effects of A23187. Chelation of extracellular calcium by 0.5 or 1 mu EGTA had no significant effect on P4 synthesis (data not shown). However, the presence of 2 mM EGTA caused an increase (about 1.8-fold, P < 0.01) in P4 production compared with that of
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3.5 r 3.0 2 t
2.5
*$
”
Nonincubated samples
Control
50 /LM
5OP 25 OH Chol
LII
3 mM EGTA 6 h Incubated
5OcLy 25 OH Chol I PM A 23187
somples
3. Effects of EGTA and calcium ionophore A23 187 in 25 OH Chol-dependent P4 secretion. The values given are means f s.d. of triplicate samples. ‘P < 0.05; ***P < 0.001 compared to control value.
Figure
untreated cells, and a plateau was obtained between 2 and 4 mu EGTA [Figure 2(b)]. Further, concomitant presence of EGTA and A23 187 led to a reversal of the inhibitory effect of the ionophore, depending on the concentration of EGTA used. While inhibition (P < 0.001) of P4 secretion was observed in the presence of 1 PM A23187 alone, stimulation (P < 0.001) appeared in the presence of 1 PM A23 187 plus 4 mM EGTA. Taken together these results suggest that extracellular calcium could play a role of negative modulator of ovine placental progesterone synthesis. We also tested the effects of EGTA and A23 187 on the conversion of 25 OH Chol to P4 (Figure 3). In this experiment 3 mu EGTA and 50 PM 25 OH Chol sign&&y stimulated basal P4 production (stimulation by EGTA 0.9-fold P < 0.05, by 25 OH Chol 1.4-fold P -=c0.001 and by 25 OH Chol plus EGTA 2.5-fold P -=c0.001). In contrast, there was a significant (P < 0.001) decrease in P4 secretion in the presence of 1 PM A23187 and also in the presence of 1 ,uM A23 187 plus 50 ,uM 25 OH Chol. Effects of PKC activators on P4 synthesis Addition of synthetic diacylglycerols OAG and DOG at 1,lO or 100 ,L~Mdid not significantly affect basal P4 secretion (data not shown). Figure 4(a) shows that a lack of effect was also observed in the presence of PMA. However, 25 OH Chol stimulated P4 secretion (P < 0.01) was reduced to basal level. Similar effects were observed in the presence of 100~~ DOG, while the non-tumor promoting phorbol ester PM, was unable to affect this secretion [Figure 4(b)]. In addition, over the same concentration range PMA also decreased EGTA-enhanced P4 secretion which remained at a basal level in the presence of 160 no PMA (Figure 5).
DISCUSSION This study demonstrates that calcium plays a key role in the regulation of P4 secretion by ovine chorionic cells. Several strands of evidence presented here suggest that calcium-
De La Llosa-Hermkr et al: Modulation ofProgesteroneSecretion by (l;zlcium
517
ES I .5 0 iI I .o : F
0.5 0
Non- Control 1.6 “M 16 no 160~ I mcubated samples PMA
d
1.6 nM 16 no 160~4
1
,
PMA
L---J 50 /LLM25 OH Chol :. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 h Incubated samples
0 Nonincubated sample
Control 100 FLM 200 no DOG
PM
100 FLM 200 “M DOG
PM
50 /.LLM25 OH Chol ~..._...__...............................,,,.............~ 6h
Incubated
samples
concentrations of PMA on basal and 25 OH Chol-dependent P4 secretion by ovine placental cells. (b) Effects ofDOG and PM on basal and 25 OH Chol-dependent P4 secretions. The values given are means t s.d. of triplicate samples. **Pi 0.01; ***P < 0.001 compared to control value.
&we
4. (a) Effect ofincreasing
dependent processes are involved in both the positive and the negative control of this secretion. First, TFP or pimozide, at concentrations similar to those which bind to calmodulin, inhibited basal P4 synthesis: it therefore seems likely that the observed inhibition results from their ability to interfere with calmodulin-dependent process. Involvement of calcium-calmodulin pathway in P4 secretion has also been demonstrated in human (Kasugai et al, 1987) and bovine (Ullmann and Reimers, 1989) placenta as well as in Leydig cells (Hall, Osawa and Mrotek, 198 1). Although the intracellular mechanism(s) underlaying the control of steroidogenesis by this pathway are not completely elucidated, it has been shown that in Leydig cells calcium-calmodulin enhanced the transport of cholesterol to the mitochondria and P450 see activity. To investigate the impact of calcium-calmodulin system on this enzymatic complex, we used 25 OH Chol. In previous work (De La Llosa-Hermier et al, 1988) we showed that addition of 25 OH Chol did not increase P4 secretion by ovine explants during the first 3 h of incubation. Our present observations of a 25 OH Chol-conversion to P4 by dispersed chorionic cells during 6 h experiments suggest that in these conditions not enough endogenous substrate is available and/or cannot be mobilized to bring about the highest rate of P4 secretion possible. Since P4 production was not significantly increased by
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0
Nanincubated samples
Control
1.6 nkl
1
16nkl
L----_-J
3 mM EGTA
:.. . . . . . . 6 h Incubated
160nu
I
PMA
.:
samples
of increasing concentrations of PMA on EGTA-stimulated P4 secretion by ovine chorionic cells. The values are means + s.d. of triplicate samples. *P < 0.05; *#P < 0.01; *++P < 0.001 compared to control value.
Figure 5. Effect
25 OH Chol when calmodulin antagonists were added concomitantly, it may be expected that the calcium-calmodulin system plays a role in regulating the content and/or the activity of P450 see enzyme. The additional possibility of treatment-induced changes in cholesterol availability remains to be investigated. Second, it would be expected from data presented here, that agents which increase intracellular calcium concentration such as ionophores, could stimulate P4 secretion by ovine chorionic cells. This was the case in Leydig cells (Lin, 1985) or granulosa cells (Wang and Leung, 1987). Surprisingly, in the present work A23187 was able to inhibit P4 production. Although this effect might be non-specific, this is unlikely, because chelation of extracellular calcium by EGTA prevented the effect of A23 187, suggesting that it was totally dependent on calcium influx from extracellular calcium. Further, EGTA stimulates progesterone secretion. Taken together these results may be interpreted on the basis of a critical pool of calcium readily exchangeable with extracelhtlar fluid, which imposes a limit on ovine placental steroidogenesis. A23187 has also been reported to inhibit basal P4 synthesis in ovine luteal cells (Conley and Ford, 1989) and large bovine luteal cells (Alila, Dowd and Corradino, 1988). However, in human (Kasugai et al, 2987) and in bovine (Ullmann and Reimers, 1989), placenta A23 187 increases P4 secretion. Hence, ionophore induces a wide variety of effects depending on the cells or species studied. From the experiments presented here, it is proposed that calcium-dependent processes may limit P4 secretion in ovine chorionic cells by decreasing P450 see activity and also by decreasing the availability of substrate. Since our results with 25 OH Chol demonstrate that P4 production is substrate limited, it may be concluded that in the presence of EGTA, substrate availability is increased. Support for action on P450 see activity was provided by the fact that the inhibitory effect of A23 187 cannot be overcome by addition of 25 OH Chol, while 25 OH Chol metabolism was enhanced by EGTA. Another hypothetical mechanism is proposed by Ghosh et al (1938) who demonstrated that during the. loss of steroidogenic function observed in hCG-
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desensitized rat luteal cells, availability of cholesterol substrate and P450 see amounts was unaffected. These authors suggested that a substance might inhibit P450 see enzyme. Whether such a substance is involved in calcium-mediated inhibition of P4 production by ovine chorionic cells, remains to be studied. As alluded to above, results obtained with ionophore and EGTA appear to conflict with those obtained in the presence of TFP or pimozide. However, this discrepancy might only be apparent. Of particular interest is the observation that, although most calmodulin-regulated enzymes bind calmodulin in the presence of calcium, calmodulin can bind phosphorylase kinase even in absence of calcium (Cohen et al, 1980). On the other hand the cell mixture used is not homogeneous and may contain steroidogenic cells which might differ in the mechanisms involved in the regulation of P4 secretion. Finally, we have shown that treatment by PKC activators, either PMA or OAG or DOG, failed to affect basal P4 synthesis. However, EGTA or 25 OH Chol failed to significantly increase basal P4 secretion in the presence of PKC activators. Thus, it appears that the highest rates of P4 production by these cells would be strong evidence of the negative effect exerted by these compounds. Since PMA and synthetic diacylglycerols are believed to affect cell functions by activating PKC, these results support the concept that activation of PKC decreases P4 production by ovine chorionic cells. An inhibitory effect of PMA on steroidogenesis has also been reported in swine granulosa (T;‘eldhuis and Demers, 1986) and ovine luteal cells (Jammes, De La Llosa-Hermier and Hermier, 1988; Wiltbank, Knickerbocker and Niswender, 1989; Conley and Ford, 1989). In agreement with these studies, our present data suggest that PKC inhibits P450 see activity in ovine chorionic cells. It is interesting to note that A23187 is able to. stimulate PKC activation (Nishizuka et al, 1984). Thus, the observed ionophore-induced decrease of P4 secretion by ovine chorionic cells might be due to activation of PKC. However, because A23 187 provokes a greater inhibition than PMA it may be concluded that, at least in part, the inhibition induced by A23 187 is independent of PKC activation. In conclusion, we have shown that ovine placenta is not autonomous in terms of P+ secretion. There is ample evidence to support the concept that calcium plays a crucial role in modulating steroidogenesis in this tissue. Furthermore calmodulin and protein kinase C may be important components of the biochemical mechanisms involved in P4 secretion. At this time we are unaware of hormones and factors which under physiological conditions control the calcium-signalling system.
ACKNOWLEDGEMENTS This study was financially supported (Fresnes, Paris).
in part by Grant 9189012 from ‘Fondation
de la Recherche
en Hormonologie’
REFERENCES Alila, H. W., Dowd, J. & Corradino, R. A. (1988) Role of calcium in progesterone synthesis by small and large bovine luteal cells. 21st Annual Meeting of the Society for the Study of Reproduction, Seattle WA, p. 55 (Abstract). Basset, J. M., Oxborrow, T. J., Smith, J. D. & Thorbum, G. D. (1969) The concentration ofprogresterone in the peripheral plasma of the pregnant ewe.Jofounzal ofEndocrikology, 45,449-457. Castagna, M., Takai, Y., Kaibuchi, K., Sane, K., Kikkawa, U. & Nishizuka, Y. (1982) Direct activation of calcium activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. Journal of Biological Chemistry, 257, 7847-785 1.
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Cohen, P., Klee, C. B., Picton, C. & Shenohkar, S. (1980) Calcium control of muscle phosphorylase kinase through the combined action of calmodulin and troponin. Annals of the New YorkAcademy of Sciences, 356, 1Sl161. Conley, A. J. & Ford, S. P. (1989) Effects of TPA, A23187 and prostaglandin Fan on progesterone synthesis by dispersed-luteal cells. BiologyofReproduction, 40, i224-1230. _ De La Llosa-Hermier. M. P.. Zonznzo. M. A.. Martal. 1. & Hermier. C. (1988) Lack of short-term modulation ’ of in vitro placental progesterone iecretion in sheep. P&nta, 9,623A3 1.’ Dufau, M. L. (1988) Endocrine regulation and communicating functions of the Leydig cell. Annual Review of Physiology,50,483-508.
Ghosh, D. K., Peegel, H., Dunham, W. R., Sands, R. H. & Menon, K. M. J. (1988) Modulation ofprogesterone synthesis and cytochrome P450 levels in rat luteal cells during human chorionic gonadotropin-induced desensitized state. Endorrinology,123,514522. Hall, P. F., Osawa, S. & Mrotek, J. (1981) The influence of calmodulm on steroid synthesis in Leydig cells from rat testis. Enducrinologv,109, 1677-1682. Jammes, H.. De La Llosa-Hermier, M. P. & Hermier, C. (1988) Contr6le negatif de la sdroidogenese du corps jaune ovine par la PMA. PathologieiBiologie,36, abstract 8. Kasuzzi. M.. Kato. H.. IrIvnma H.. Kato. M.. Ninatmwa. T. & Tomoda Y. (1987) The role of Ca2+ and adeiosine 3’,5 ‘-monophoiphate’in the regtdatidn of progesterone production’by human’placental tissue.joburnal of ClinicalEndocrinologyand Metabolism, 65, 122-126. Kraft, A. S. & Anderson, W. B. (1983) Phorbol esters increase the amount of calcium Ca2+ phospholipiddependent protein kinase associated with plasma membrane. Nature, 301,621-623. Lin, T. (1985) The role of calcium/phospholipid-dependent protein kinase in Leydig cell steroidogenesis. Endocrinology,117,119-126. Martal, J. & Djiane, J. (1977) The production of chorionic somatomammotrophm in sheep.3oumal OfReproduction and Fertility, 49,285-289.
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Thorburn, G. D., Challis, J. R. G. SKRobinson, J. S. (1977) Endocrine control of parturition. In Biologyof the Uterus (Ed.), Wynn, R. M. pp. 653-732. New York and London: Plenum Press. Toaff, M. E., Schleyer, H. & Strauss III, J. F., (1982) Metabolism of 2%hydrozycholesterol by rat luteal _ . mitochondria and dispersed cells. Ena&ino&y, lI1; 1785-1790. Ulhnann. M. B. 8z Reimers. T. 1. (1989) Prozesterone nroduction bv binucleate troohoblastic cells of cows. Jounzal’ofReproduction and Ft&ili&, j7, Snppl. i73-179. * Veldhuis, J. D. & Demers, L. M. (1986) An inhibitory role for the protein kinase C pathway in ovarian steroidogenesis. Studies with cultured swine granulosa cells. BiochemicalJournal, 239,505-511. Wang, J. & Leung, P. C. K. (1987) Role of protein kinase C in luteinizing hormone-releasing hormone (LHRH)stimulated progesterone production in rat granulosa cells. BiochemicalBiophysicalResearch Communications,146, 939-944. Wiltbank, M. C., Knickerbocker, J. J. & Niswender, G. D. (1989) Regulation of the corpus luteum by protein ldnase C. I. Phosphorylation activity and steroidogenic action in large and small ovine luteal cells. Eiologv of Reproduction,40,11941200.
Wooding, F. B. P. (1982) The role of the binucleate cell in ruminant placental structure.~oumalofReprodu&n Fertility, 31,31-39.
and