C. Hermier

Evidence for modulation of progesterone secretion by calcium and protein kinase C activators in ovine chorionic cells

The hypothesis that calcium-dependent mechanisms may be involved in regulating ovine placental steroidogenesis was investigated using chorionic cells isolated by enzymatic digestion. Treatment of the cells with the calmodulin antagonist trifluoperazine (TFP) or pimozide caused a dose-related inhibition of progesterone (P4) production by 80 percent (P less than 0.001) at 40 microM TFP and 56 per cent (P less than 0.001) at 10 microM pimozide. Moreover, the conversion of 25 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 and completely blocked it at 1 microM (P less than 0.001) and remains absent even in the presence of 25 OH Chol. In contrast, EGTA increased P4 secretion (P less than 0.01). Further, in the presence of 3 mM EGTA the inhibitory effect of 1 microM A23187 was fully reversed. Taken together these results suggest that extracellular calcium could play a role of negative modulation of P4 secretion in these cells. The possible involvement of protein kinase C (PKC) was tested using tumor-promoting phorbol ester (PMA) or permeant diacylglycerols (OAG or DOG). These compounds were unable to modify basal P4 secretion but reduced 25 OH Chol stimulated secretion to basal level. The phorbol ester that was unable to activate PKC had no effect on the metabolism of 25 OH chol. Thus, PMA and diacylglycerol effects are 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 of P4 secretion by ovine placenta. Our results also suggest a role for calmodulin and PKC pathways in modulating this secretion.

Lack of short-term modulation of in vitro placental progesterone secretion in sheep

Experiments were performed in order to determine whether progesterone secretion in the ovine placenta can be short-term regulated. There was an increase in progesterone content per unit weight in ovine fetal cotyledons as gestation progressed: 17.0 +/- 4.7 ng/100 mg of wet tissue in ewes between 40 and 54 days of pregnancy (n = 7) and 70.7 +/- 18.8 (n = 9) between 100 and 118 days. At all stages of pregnancy, neither progesterone nor 20 alpha-dihydroprogesterone synthesis were significantly affected when fetal cotyledons were incubated for 3 h in the presence of LH, 8-Br-cAMP, GnRH agonist or GnRH antagonist. Addition of pregnenolone to the incubation medium increased progesterone secretion in a dose-dependent manner while addition of 25-hydroxycholesterol did not. These results suggest that the existent (basal) synthesis of progesterone reflects the maximal capacity of steroidogenesis through the cholesterol side-chain-cleavage system. In the presence of these precursors, LH, 8-Br-cAMP, the phorbol ester derivative PMA and calcium ionophore A23187 were not able to modify progesterone or 20 alpha-dihydroprogesterone synthesis. These results also suggest that LH or GnRH and the two signal mechanisms involved in their action, i.e. cAMP and Ca2+ sensitive-inositol phospholipid-dependent mechanisms are not implicated in the short-term regulation of progesterone synthesis in the ovine placenta.

In vitro effect of prolactin, prostaglandin F2α and cycloheximide on 20α-dihydro-progesterone synthesis in pseudo-pregnant rat ovaries

Abstract Experiments were designed to study the effects of prostaglandin F2α and prolactin in the modulation of steroidogenic response of the corpus luteum. Pseudo-pregnant rat ovaries, obtained by treatment with PMSG and HCG, were incubated or perifused in the presence of these compounds and the 20α-dihydro-progesterone (20α-DH-P) secretion and 20α-hydroxysteroid-dehydrogenase (20α-OH-SDH) activity estimated. The 20α-DH-P synthesis during pseudo-pregnancy in the rat increased for 21 days after HCG treatment and decreased thereafter. PG F2α increases 20α-DH-P secretion and 20α-OH-SDH activity whereas prolactin inhibits the amount of 20α-DH-P released and the 20α-OH-SDH activity. Cycloheximide blocked the response to PG F2α and which suggests that protein synthesis plays a part in this mechanism. Prostaglandin and prolactin failed to modify the 20α-OH-SDH activity when added directly to the “105,000 g” fraction isolated from these ovaries.

Activation of phospholiphase C by different effectors in rat placental cells

The present communication documents the accumulation of inositol phosphates in rat placental cells by fluoride as well as by vanadate. These findings suggest the existence of the phosphoinositide pathway and its modulation by a G-protein. A concomitant action of fluoride on phosphoinositide breakdown was also observed. As is often the case in intact cells from different organs, protein kinase C exerts a feedback regulatory control on this signalling system. Gonadotropin-releasing hormone (GnRH) also stimulated the accumulation of inositol phosphates in cultured cells but no effect could be detected in freshly isolated cells. Therefore, the phosphoinositide pathway seems to be involved in the mechanism of action of GnRH in rat placental cells.

Studies of the binding activity to different gonadal receptors of ovine luteinizing hormone (LH) after chemical modification of lysine residues.

Abstract Several LH derivatives obtained by modification of the lysine residues were studied by the radioligand receptor assay using either corpora lutea homogenates or testis homogenates and tritiated methylated LH. The binding activity found was very similar to the biological activity as determined in the ovarian ascorbic acid depletion test, although some minor differences were observed. The binding activity of the methylated derivatives was no different from that of native LH, whereas the other alkylated derivatives were less active or inactive. As expected, carbamylated LH was inactive and the activity of guanidinated LH was considerably reduced. Some differences were observed in the binding affinities of these derivatives when comparing corpora lutea with testicular homogenates.

Concurrent LH and forskolin action on adenylate cyclase activation and progesterone synthesis in corpora lutea from pregnant ewes.

The present communication documents LH- and forskolin-induced activation of adenylate cyclase (AC) system and progesterone synthesis in corpora lutea from pregnant ewes. The activation of AC in plasma membranes by LH or forskolin was amplified by Gpp(NH)p. These results suggest that regulatory nucleotide component (Ns) of the AC complex is required for forskolin. Simultaneous addition of maximal concentrations of forskolin (10(-4) M), Gpp(NH)p (10(-4) M) and LH (10(-7) M) led to greater than additive (i.e. synergistic) responses: the experimental value was 4.71 +/- 0.19 nmoles cAMP/mg of membrane protein, whereas the theoretical additive effect was 3.17 +/- 0.10 nmoles/mg of membrane protein (p less than 0.001). These data reveal that more Ns or C component is being activated in these cells when combined treatments with these agents are applied. In intact cells maximum stimulatory concentrations of forskolin or LH caused similar increase in progesterone production with similar time courses. In striking contrast, the exposure of the luteal cells to LH and forskolin simultaneously led to a decrease in progesterone synthesis as early as 1h30 (40%, p less than 0.001). Thus, the synergism observed between LH and forskolin on the stimulation of plasma membranes AC activity did not occur in steroidogenesis. The AC responses in crude plasma membranes form these cells to different stimulants were enhanced (i.e. 15%, p less than 0.2 for Gpp(NH)p, 33%, p less than 0.01 for LH plus Gpp(NH)p and 52%, p less than 0.01 for forskolin). These findings suggest that an early desensitization of the AC system cannot explain the impaired steroidogenic response observed.

Etude in vitro de la synthese de la progesterone dans le corps jaune cyclique humain: Role de la prostaglandine F2α

When HCG and PG F2α are added into the medium at their “maximal effective dose” the effect is supra‐maximal. In our experimental procedure, acetylsalicylic acid does not inhibit the synthesis of progesterone stimulated by LH. According to these results it seems that PG F2α is not the necessary membrane mediator between the hormonal‐receptor and the adenylcyclase system.

20-α-Hydroxysteroid dehydrogenase from pseudopregnant rat ovary: Obtention and characterization of a monoclonal antibody against the enzyme activity

The enzyme 20-alpha-hydroxysteroid dehydrogenase (20-alpha-HSD) was purified from pseudopregnant rat ovaries and used as antigen for the development of a monoclonal antibody by the hybridoma technique. Spleen cells of BALB/c mice immunized with purified 20-alpha-HSD were fused with SP2/0 mouse myeloma cells. Among the colonies of hybrid cells, one (designated mAb-HSD 11) was found to be secreting antibodies (IgM) able to inhibit 20-alpha-HSD activity. The antibody-secreting hybridome was amplified by ascitic fluid production and the monoclonal antibody purified by Bakerbond ABx procedure. Purified mAb-HSD 11 was able to inhibit 20-alpha-HSD activity in a dose-dependent manner. Studies of Michaelis constants of 20-alpha-HSD indicate that this monoclonal antibody increases the Km for 20-alpha-dihydroprogesterone and decreases the Vmax.

Adenylate cyclase stimulation and luteinizing hormone-receptor interaction in plasma membranes from rat testicular interstitial cells in relation to the chemical structure of the hormone. Role of Mg2+.

To understand more closely the structural requirements of the LH molecule necessary to stimulate adenylate cyclase, we studied the modulation of this enzyme in partially purified plasma membranes prepared from isolated interstitial cells of rat testis submitted to oLH and to some oLH derivatives and natural analogues. The role of Mg2+ was also investigated in relation to the structural modifications of oLH. Some new facts appeared in this study: 1. Methyl oLH, which exhibited the same ability as native oLH to stimulate cAMP accumulation and steroidogenesis in isolated cells, cannot induce the same level of maximal stimulation of adenylate cyclase as native oLH in plasma membranes. This phenomenon is related to the Mg2+ concentration, and the differences between maximal activation induced by methyl oLH and oLH were more apparent at a free Mg2+ concentration of 3.3 mmol/l than at lower concentrations. The maximal activity (in terms of native oLH) of other alkyl derivatives, such as ethyl or isopropyl oLH, on the contrary, was similar in isolated plasma membranes and in intact cells suggesting that the differential behaviour of the membranes specifically concerns the methyl derivative. 2. Guanidyl oLH and guanidyl porcine LH, which were able to induce cAMP accumulation in intact cells, did not exhibit any stimulating activity in plasma membranes. 3. Among the natural analogues, hCG and pLH are distinguished by a lower maximal activity (by comparison with oLH) particularly at high Mg2+ concentration. This work shows that changes in the LH structure have an impact not only on the parameters of the adenylate cyclase complex but also on the transduction of the hormone signal and its modulation by Mg2+.

Action of nitroguanidinated luteinizing hormone on different rat gonadal tissues

The biological activities of nitroguanidinated derivatives prepared from ovine or porcine luteinizing hormone were investigated using rat Leydig cells and pseudopregnant rat ovaries. In these tissues nitroguanidyl ovine luteinizing hormone (NGoLH) or nitroguanidyl porcine luteinizing hormone (NGpLH) were unable to stimulate adenylate cyclase or steroidogenesis but were able to inhibit the binding of ovine or porcine native LH to their specific receptors. When added to incubations of isolated Leydig cells or pseudopregnant ovary slices, NGoLH as well as NGpLH inhibited the stimulating action of native LH on adenylate cyclase or steroidogenesis. However, these derivatives had no inhibiting action on the stimulation of adenylate cyclase and steroidogenesis induced in the Leydig cells by choleratoxin or on the stimulation of testosterone production induced by 8-bromo-cyclic AMP. Since NGpLH (which does not contain lysine residues or free alpha-amino groups in the beta-subunit) exhibits the same antagonist action as NGoLH, we conclude that the nitroguanidination of the alpha-subunit is sufficient to endow the derivative with antihormone properties.