Interferon-tau upregulates prolactin receptor mRNA in the ovine endometrium during the peri-implantation period.

Our objective was to determine the effect of ovine interferon-tau (IFN-tau) on prolactin receptor (PRL-R) gene expression in the ovine endometrium. IFN-tau is an embryonic cytokine which, via its paracrine anti-luteolytic activity, plays a critical role in maternal recognition of pregnancy in ruminants. Using ribonuclease protection assay procedures, we compared endometrial PRL-R mRNA levels in ewes that were intrauterine injected with either 2 mg bovine serum albumin or 2 mg recombinant ovine IFN-tau on day 10 of the oestrous cycle (day 0 = day of oestrus). IFN treatment significantly increased the abundance of both the long and short forms of PRL-R mRNA in the ovine uterus, but had no effect on the long:short form ratio. In situ hybridization experiments revealed that the increase in abundance of PRL-R mRNA in the uterus was localized to the glandular compartment of the endometrium. In pregnant ewes, a similar increase in PRL-R mRNA abundance was found to occur in ovine endometrium on days 14-15 post conception. Collectively, these data provided strong evidence that IFN-tau modulates the level of lactogenic hormone receptor mRNA in the ovine uterus. Whether the effect of IFN-tau on PRL-R expression is mediated directly or influenced, at least in part, by progesterone remains to be elucidated.

[Trophoblastic interferons and embryonal immune tolerance]. / Les interférons trophoblastiques et la tolérance immunologique embryonnaire.

Current evidence support the hypothesis that trophoblast interferons play a key role in preventing maternal immunologal rejection of the embryonic semi-allograft. The information of this review is divided in two sections. In the first section we described molecular and biological characteristics of type I (alpha, beta, omega, tau and spl) and type II (gamma) interferons. In the second section we emphasize studies on immunoendocrine functions of IFN-tau (oTP-1 or trophoblastins) in the network of cytokines and hormonal environment at the uterine embryonic interface.

[Cytokines and the corpus luteum]. / Cytokines et corps jaune.

This minireview present main findings concerning the contribution of cytokines to the regulation of some key processes of luteal functions. Data concerning the preovulatory follicles invasion by white blood cells and the migration of macrophages, granulocytes and T lymphocytes into corpus luteum suggest that local secretion of regulatory cytokines may be involved in regulating corpus luteum formation and demise as well its maintenance in early pregnancy. Several lines of evidence indicate that the pro-inflammatory cytokines such as IL-1, IL-6 and TNF-alpha modulate the steroidogenic changes which take place during luteinization. For instance, an inhibition of E2 biosynthesis is evidenced in granulosa cells in human or porcine species with IL-1, in rat with TNF-a and in bovine with IL-6. Moreover, IL-1 stimulates P4 production but to a much lower extent than LH, and PGE2 synthesis by rat thecal cells. The potential relevance of pro-inflammatory cytokines in the mechanisms controlling luteolysis is suggested by the ability of IL-1 and TNF-alpha to decrease both P4 production and the survival of bovine luteal cells. As opposed to ruminants, TNF-alpha has no effect in human luteal cells but potentiates the decrease of P4 secretion induced by IFN-gamma. Finally, data regarding the participation of trophoblast interferons in the mechanisms for maintaining the corpus luteum at the establishment of pregnancy are now available in ruminants. From these observations and others, we can consider that cytokines are involved in the regulation of the corpus luteum function.

Potential role for arachidonic acid and eicosanoids in modulating progesterone secretion by ovine chorionic cells.

The present study was conducted to investigate whether arachidonic acid and its metabolites can modulate progesterone (P4) secretion in ovine chorionic cells. At concentrations of 7.5 mumol/l and 12.5 mumol/l, arachidonic acid caused an increase of basal P4 secretion (about 1.8-fold (p < 0.01) and 2.5-fold (p < 0.001), respectively, over control). Such a stimulatory effect was suppressed when the concentration of arachidonic acid attained 25 mumol/l, and at 50 mumol/l the fatty acid led to a decline of basal P4 synthesis (about 35%, p < 0.01). Phospholipase A2 (PLA2) and melittin had a similar dual effect to that observed when arachidonic acid was added exogenously. In contrast, eicosatrienoic acid (a closely related fatty acid) did not stimulate P4 secretion but inhibited it at a concentration of 50 mumol/l (about 40% inhibition, p < 0.01). The possible involvement of calcium on the effects of arachidonic acid was explored. Interestingly, 3 mmol/l ethylene glycol bis(beta-aminoethyl ether)-N,N,N,N'-tetraacetic acid (EGTA) and 10 mumol/l 8-N,N-diethylamino-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) further enhanced the steroidogenic effect of 12.5 mumol/l arachidonic acid (p < 0.05 and p < 0.01 vs the corresponding value in the absence of EGTA or TMB-8, respectively). In contrast, these agents failed to modify P4 secretion observed in the presence of 50 mumol/l arachidonic acid. We also tested the effect of inhibition of arachidonic acid metabolism via cyclooxygenase and lipoxygenase pathways. Indomethacin (10 mumol/l) failed to block the effects of arachidonic acid, but nordihydroguaiaretic acid (10 mumol/l) prevented the stimulatory action of this fatty acid.(ABSTRACT TRUNCATED AT 250 WORDS)

20-alpha-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.

Activation of phospholipase 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.

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.

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+.

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.

A comparative study of adenylate cyclase activity and progesterone synthesis in ovine corpora lutea stimulated by different chemical derivatives or natural analogs of ovine LH.

The adenylate cyclase activation by ovine native LH, natural analogs (porcine LH, hCG) and chemical derivatives of LH (methylated, ethylated, isopropylated, guanidinated) was studied in purified plasma membranes of ovine corpora lutea, including the regulatory effects of guanyl 5'-yl imidophosphate (Gpp(NH)p) and Mg2+. The Ka app. for native LH (about 15 nM) was independent of Gpp(NH)p and Mg2+. Similar maximal activation of the enzyme was obtained by using ovine LH or natural analogs, but differences were remarked concerning the Ka app. values of these hormones. Porcine LH was equipotent with ovine LH; on the contrary, hCG exhibited a lower Ka app. value (3 nM). All chemical derivatives (Me-LH, Et-LH, Iso-LH and Gu-LH) exhibited Ka app. higher than native (about 2- to 4-fold), but similar maximal activation. No modification was observed in the regulatory effects of Gpp(NH)p and Mg2+ on the adenylate cyclase activation as a consequence of structural modifications of the hormone. A comparison of the steroidogenic activity on intact luteal cells and the adenylate cyclase activation ability on purified plasma membranes of the derivatives mentioned above evidenced some interesting discrepancies. The drop in adenylate cyclase activation potency of Me-LH was not reflected in its steroidogenic activity (Me-LH was equipotent with native LH); on the contrary, the capacity of Gu-LH to stimulate adenylate cyclase was not so much decreased as was its steroidogenic potency which was almost abolished.

Properties of an LH-stimulable adenylate cyclase in plasma membranes of interstitial cells from rat testis.

We studied the effect of guanyl nucleotides, divalent cations and luteinizing hormone (LH) on the regulation of adenylate cyclase (AC) in partially purified plasma membranes obtained from isolated interstitial cells of the rat testis. AC was activated to different degrees by guanosine triphosphate (GTP) and GMP-P(NH)P; the latter was about 10 times more active than the former. Enzyme activation by GTP was biphasic; the nucleotide was rapidly hydrolysed by membrane preparation. Activation by GMP-P(NH)P was hysteretic, requiring about 20-30 min to reach steady state; this lag-time was not dependent on nucleotide concentration. GDP beta S did not stimulate AC activity. Delayed addition of GDP beta S to a GMP-P(NH)P-stimulated enzyme at 17 min resulted in a drop of AC activity although the activity 40 min later was higher than that obtained by mixing both nucleotides at the time the reaction was initiated. This result was incompatible with the formation of a truly irreversible, active form of the enzyme in the presence of GMP-P(NH)P. The effect of LH on AC depended on guanine nucleotides and Mg2+. LH and GMP-P(NH)P acted synergically. Dose-response curves showed that apparent LH affinity was not modified by the presence of GMP-P(NH)P. LH accelerated the slow rate of activation of GMP-P(NH)P. The stimulation of AC by LH was closely dependent on Mg2+ concentrations; LH diminished the apparent Mg2+ requirement. Plasma membranes from rat testicular interstitial cells are an excellent model for the study of AC regulation by LH.

[Steroidogenic action of 2 analogs of ovine LH on cells isolated from ovine corpus luteum]. / Activité stéroïdogénique de deux analogues de la LH ovine sur cellules isolées de corps jaunes ovins.

Lysine residues appear to play an important role in the biological activity of luteinizing hormone or lutropin (LH). Some derivatives obtained by chemical modification such as N-methylated LH exhibit the same hormonal activity than LH in the different steps of the mechanism of steroidogenesis. Some others, on the contrary, preserve the hormonal activity only at some steps. In this work was investigated the action of ovine LH and some derivatives on isolated cells prepared from ovine corpora lutea. Guanidinated LH (which is able to bind to LH receptors in Leydig cells but whose steroidogenic potency is very low) exhibits no binding or steroidogenic activity in the female (sheep or rat). As a consequence guanidyl LH can act as an inhibitor of LH action in the male (Leydig cells).

Effects of antimicrotubular agents in cAMP production and in steroidogenic response of isolated rat Leydig cells.

In dispersed rat Leydig cells, colchicine was found to stimulate basal cAMP production and testosterone secretion in a dose and time-dependent manner, but to a lesser extent than LH. However, these drugs are unable to stimulate adenylate cyclase activity in plasma membranes isolated from these cells. The amount of testosterone secreted at 150 min under the influence of colchicine and LH added simultaneously was not different from the amount produced during stimulation by LH alone. It is only after exposure of the cells for 1 hr to colchicine that the accumulation of cAMP in response to LH was inhibited; furthermore, both intracellular and medium testosterone accumulation in response to the hormone were reduced. Similar effects were observed with two other alkaloids, vinblastine and podophyllotoxin. The three drugs also inhibited the stimulation of testosterone secretion by 8-Br-cAMP or choleratoxin. These studies suggest that the state of microtubule polymerization and/or tubulin can influence the process of steroidogenesis in rat Leydig cells.

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.

Action of some luteinizing hormone derivatives in ovaries from pseudopregnant rats: dissimilarities between their activities on this organ and on Leydig cells.

Biological activities of several derivatives of ovine LH obtained by chemical modification of the amino groups were investigated using ovaries from pseudopregnant rats. Binding-inhibition activities and steroidogenic potencies of ethylated, isopropylated and guanidinated LH were in good agreement, whereas adenylate cyclase activities were relatively greater. When compared with previous results on binding-inhibition activities and steroidogenic potencies using isolated rat Leydig cells, the ovaries from pseudopregnant rats appeared to be more discriminating. Ethylated and isopropylated derivatives exhibited lower binding-inhibition activities and steroidogenic potencies in female gonads. This difference was particularly evident in the case of guanidinated LH which exhibited a very low binding-inhibition activity and consequently was unable to act as an inhibitor of the action of LH on the ovaries. Guanidinated porcine LH (in which all the lysine residues of the alpha-subunit were transformed into homoarginine, without modification of the beta-subunit which does not contain lysine) showed similar biological activities to guanidinated ovine LH in the isolated Leydig cells as well as in pseudopregnant ovaries. It can, consequently, act as an inhibitor of LH action on Leydig cells but not on the ovary of the pseudopregnant rat. Thus, the inhibitory properties of this derivative can be ascribed to the modification introduced in the alpha-subunit.

Inhibitory effect of ovine and human placental lactogens on progesterone catabolism in luteinized rat ovaries in vitro.

Pseudopregnant rat ovaries, obtained by PMSG and hCG treatment, were incubated or perfused. Progesterone and 20 alpha-dihydroprogesterone (20 alpha-DHP) secretion and synthesis as well as 20 alpha-hydroxysteroid dehydrogenase (20 alpha-SDH) activities were determined by radioimmunoassays. Ovine and human chorionic somatomammotrophin (oCS; hCS) and ovine prolactin (10(-8) M) significantly (P less than 0.001) depressed 20 alpha-DHP ovarian contents (49, 64 and 69 per cent, respectively) and 20 alpha-DHP release (49, 55 and 55 per cent, respectively) in comparison with those of controls without hormones. The activity of 20 alpha-SDH was also inhibited by these three hormones (59, 68 and 56 per cent, respectively). The kinetics of 20 alpha-DHP release, studied by perfusion or incubation, showed a maximum inhibition from the first hour onwards with all lactogenic hormones. The fall in 20 alpha-DHP content of luteinized ovarian tissue coincided with a sharp rise in the progesterone/20 alpha-DHP ratio, from 0.5 in the controls to 1.5 after lactogenic hormone treatment. This study in vitro showed that two placental lactogens of sheep or human origin exhibit an inhibitory effect on 20 alpha-SDH activity analogous to that of prolactin. It is suggested that these placental hormones may be involved in the regulation of luteal and placental progesterone metabolism during pregnancy.

LH receptors in ovine corpora lutea in relation to various physiological states and effects PGF-2 alpha on LH-induced steroidogenesis in vitro.

The LH binding properties (determined using tritiated methylated LH) and the in-vitro steroidogenic activity of CL from ewes in the oestrous cycle or early pregnancy (Day 18) were compared. No significant alteration in the Kd values was observed. However, the number of sites was maximal at Day 10 of the cycle and in early pregnant animals which had not been pregnant for at least 3 months (dry ewes). Non-lactating or suckling ewes had half the numbers of binding sites. The increase of the number of receptor sites was accompanied by a steroidogenic response at lower LH concentration. During incubation or superfusion for 5 h, a refractoriness to LH stimulation appeared after 1 h with high LH concentrations and after 3 h with low concentrations. The opposite effect of the addition of indomethacin or PGF-2 alpha suggests the intervention of PGs in this phenomenon.

A comparative study of the action of several lutropin derivatives on rat Leydig cells.

Rat intestinal cells prepared from testes were incubated in the presence of different lutropin derivatives obtained by chemical modification of the amino groups. The cAMP accumulation and the testosterone biosynthesis were determined in the cell homogenates. Binding determinations were carried out by a radioligand receptor assay using tritiated methylated lutropin. The binding activities--relative to native LH--of three different derivatives obtained by reductive alkylation (methylated, ethylated and isopropylated LH) were in good agreement with the relative potencies assessed by their capacity to stimulate cAMP and testosterone production. Guanidinated LH (11-NH2 groups modified) exhibited a binding activity and a relative potency relatively high with regard to cAMP accumulation (as compared with that of native LH). Its steroidogenic potency, however, was very low. When Leydig cells were incubated in the presence of native and guanidinated LH, the testosterone production was similar to that induced by the derivative alone, indicating that the derivative exerted a competitive inhibitory action preventing the stimulation of steroidogenesis by native LH. These results suggest that a guanidinated derivative is able to bind to the LH receptor and the complex so formed is able to be coupled with an adenylate cyclase pool (or cAMP compartment) which is not connected with the steroidogenic pathway.