Rat gonadotropin-releasing hormone receptor expressed in insect cells induces activation of adenylyl cyclase.

Increasing evidence exist that multiple G proteins mediate the effects of gonadotropin-releasing hormone (GnRH) on the synthesis and release of pituitary gonadotropins. In the present study, we have expressed the rat GnRH receptor (GnRH-R) in insect cells, by infection with a recombinant baculovirus. Under the conditions used, insect cells expressed, 48 h post-infection, a maximum of 7800 +/- 650 receptors/cell which bound GnRH agonist [D-Trp6]GnRH with a Kd = 0.52 +/- 0.06 nM indicating characteristics similar to those of the natural receptor. No binding was observed in non-infected cells or cells infected with wild-type baculovirus. In presence of GnRH, GnRH-R expressing cells elicited a time- and dose-dependent production of inositol trisphosphate, with a maximum level reached within 30 min and an EC50 = 5 nM. These recombinant insect cells also produced cAMP in response to GnRH. However, in contrast to other heterologous systems, or rat pituitary gonadotropes wherein GnRH induced a weak and delayed elevation of cAMP, in insect cells the rise of cAMP was comparatively rapid, attaining a maximum level after 2 h, and the EC50 was 5 nM. Finally, a clear activation of adenylyl cyclase (AC) in response to GnRH was shown for the first time by measuring the conversion of [alpha-32P]ATP into labeled cAMP, using membrane preparations from GnRH-R expressing insect cells. These data demonstrate that rat GnRH-R has the potential for dual coupling to both phosphoinositidase C and AC and suggest a major influence of the host cell for this coupling and/or its expression, probably in relation with the G protein repertoire and preference. This notion could be extended to several target cells other than pituitary gonadotropes that normally express the GnRH-R in mammals, including hippocampal, Leydig, granulosa, placental and GnRH-secreting hypothalamic cells.

Modulation of membrane fluidity and lipidic metabolism in transformed rat fibroblasts induced by the sesquiterpenic hormone farnesylacetone.

Farnesylacetone is a natural terpene extracted from androgenic glands of the crustacean Carcinus maenas and is capable of inhibiting proliferation, notably in transformed mammalian cells. Flow cytometry with three lipophilic probes, diphenylhexatriene, trimethylammonium-diphenylhexatriene, and Nile red, has revealed modifications of the lipidic metabolism in transformed FR3T3-mTT4 rat fibroblasts treated by farnesylacetone, including changes in membrane fluidity. Farnesylacetone strongly increased the number of neutral lipidic droplets in the cytoplasm. Moreover, after prolonged terpene treatment, the membrane fraction of cells contained a substantial level of triglycerides. Farnesylacetone provoked an immediate but transitory increase in membrane fluidity of the cell membrane. The change in measured lipid fluidity appears to be due to these triglycerides rather than to the phospholipids.

Modulation of fibroblast response to maitotoxin along the cell division cycle.

Maitotoxin (MTX) induces an increase of [Ca2+]i and of phosphoinositide breakdown in various cell types. The [Ca2+]i increase followed with fluorescent probes on cell suspensions has been described as slow and lasting, in contrast to the "signal" induced by calcium ionophores such as ionomycin. MTX effects have been studied on two fibroblastic cell lines, BHK21 C13 and FR 3T3, synchronized by serum deprivation treatment performed in an isoleucine-free medium for BHK21 C13 cells. In BHK21 C13 cells, flow cytometry analysis showed that two stages, G1/S and G2/M, were particularly susceptible to MTX treatment. Scanning laser cytometry demonstrated that calcium response of FR 3T3 fibroblasts followed with Indo-1 varied during the cell division cycle. The [Ca2+]i increase was almost always vertical, but its delay after MTX addition lasted from zero (S and G2/M transition) to 10-20 min (G1) or more (G2). No [Ca2+]i change could be detected during mitosis. The [Ca2+]i response at the S phase was biphasic. These observations suggest that (1) the lasting response described in the literature represents a global cell population effect, and (2) cells are more sensitive to MTX at specific stages of the cell division cycle, which could correspond to periods when calcium signals have been detected in different cell types.

Farnesylacetone, a sesquiterpenic hormone of Crustacea, inhibits electron transport in isolated rat liver mitochondria.

Farnesylacetone (C18 H30 0) is a male hormone extracted from the androgenic gland of crab, Carcinus maenas. Appropriate enzymatic assays, as well as spectrophotometric studies, indicate that micromolar concentrations of farnesylacetone interact with the electron transport pathway of rat liver mitochondria. By the use of artificial electron donors and electron acceptors, it is shown that farnesylacetone immediately inhibits the electron transfer within complex I (NADH ubiquinone reductase activity) and complex II (succinate ubiquinone reductase activity). It is proposed that farneylacetone could interact with these two complexes of the respiratory chain at the level of the iron-sulfur centers implicated in the dehydrogenase activities. These observations are compared with the results obtained with terpenic molecules which interact with mitochondrial respiration.

Comparative effect of farnesylacetone on macromolecular synthesis in gonads of crustaceans.

Farnesylacetone, a molecule isolated from the androgenic glands of Carcinus maenas, inhibits vitellogenesis in the ovaries. Here the effect of farnesylacetone on protein and RNA synthesis in cultured organs is described. Both leucine and uridine incorporation in ovaries were inhibited with low concentration of farnesylacetone. The action was gonad specific and not species specific. It was observable in the presence of cycloheximide but not in the presence of actinomycin. This result plus the fact that inhibition of protein synthesis paralleled that of RNA suggest that farnesylacetone modifies transcription and only indirectly affects protein synthesis.

Ecdysteroids during the third larval instar in 1(3)ecd-1ts, a temperature-sensitive mutant of Drosophila melanogaster.

The temperature-sensitive 1(3)ecd-1ts mutation (A. Garen, L. Kauvar, and J.A. Lepesant (1977). Proc. Natl. Acad. Sci USA 74, 5099-5103.) has been used in several laboratories to obtain Drosophila larvae deprived of moulting hormone. The development of mutants and controls during the third larval instar at permissive (20 degrees C) and restrictive temperatures (29 degrees C) was compared. Pupariation was inhibited when larvae were shifted to the restrictive temperature immediately at the second moult. The permanent larvae obtained remained active, did not leave the food, and reached a maximum weight superior to the weight of controls. Ecdysteroids were studied during the third larval instar by HPLC analysis and radioimmunoassays. A careful synchronization of the larvae at the second moult enabled the confirmation that at least one ecdysteroid peak occurs during the third larval instar, prior to the wandering stage in controls (20 or 29 degrees C). Ecdysone was then the predominant moulting hormone, whereas 20-hydroxyecdysone was the main ecdysteroid at the time of pupariation. Low levels of ecdysteroid were measured in mutant larvae shifted to 29 degrees C immediately at the second moult but larvae completely deprived of immunoreactive material were never observed. Nearly normal levels of ecdysteroids appeared at 27.5 degrees C. Feeding ecd-1 larvae maintained at restrictive temperature on 20-hydroxyecdysone-yeast mixture for 16 hr triggered abortive pupariation. Ecdysteroid levels were measured after the return of the larvae to the standard medium; normal levels were restored 24 hr later. The mutant ecd-1 appears to present interesting opportunities for the detailed study of the hormonal induction of a developmental process during the third larval instar.(ABSTRACT TRUNCATED AT 250 WORDS)

The initiation of pupariation in Drosophila: dependence on growth of the imaginal discs.

Regeneration was induced in the imaginal discs in situ following lesions caused by heat-sensitive cell-lethal mutations. A clonal analysis of this event demonstrated that the subsequent delay in pupariation was correlated with the amount of extra growth that occurred during the regeneration. Pupariation of heat-treated gynandromorphs bearing the mutations was also retarded, and the duration of larval development increased with greater amounts of mutant tissue, it was therefore correlated with the extent of the lesions in the imaginal discs. Elimination of entire imaginal discs, or the presence of very small amounts of lethal tissue, did not result in prolonged larval life.

[Inhibition of biological methylations by t,t-farnesylacetone, a constituant of the androgenic gland of the male crab, Carcinus maenas]. / Inhibition de méthylations biologiques par ia t,t-farnésylacétone, constituant de la glande androgène du crabe mâle Carcinus maenas.

t, t-farnesylacetone 1 and hexahydrofarnesylacetone 2 have been previously identified in extracts from the androgenic gland of the male Crab Carcinus maenas. These compounds inhibit in vitro the methylation of E. coli B tRNA and of Calf thymus histones with S-adenosylmethionine methyl-14C as methyl donor and methylases from Crab testis. Rat liver or a 1-adenine methylase from a Mouse plasmocytoma (1 is approximately 200 times more active than 2).

[Methylation of tRNA in Carcinus maenas gonadal cultures: inhibition by purified androgenic gland extract]. / Méthylation du tRNA dans des cultures de gonades de Carcinus maenas: inhibition par un extrait purifié des glandes androgènes.

Subcultures of ovaries and testis of the crab Carcinus maenas have been performed in the presence of L-[Me-14C]methionine. Introduction in the medium of a chromatographically-purified liposoluble fraction from the androgenic glands of the same animal inhibits the biological methylation of the tRNA of the ovaries by 62%. The inhibition of methylation of five individual bases varies from 45% to 84%. No inhibition of tRNA methylation is observed under the same conditions with testis subcultures.

6,10,14-Trimethylpentadecan-2-one and 6,10,14-trimethyl-5-trans, 9-trans, 13-pentadecatrien-2-one from the androgenic glands of the male crab Carcinus maenas.

2 C18 isoprenoid ketones, hexahydrofarnesylacetone (1) and farnesylacetone (2) have been identified for the first time in lipid extracts from the androgenic glands of the male crab Carcinus maenas, using coupled gas chromatography-mass spectrometry. The 2 compounds prepared by synthesis, are biologically active, inhibiting the incorporation of 3H-leucine in Crustaceans ovaries subcultures.

[Determination of the ecdysone level in the molting cycle of the crab, Carcinus moenas: comparison between healthy individuals and those parasitized by Sacculina carcini]. / Détermination du taux d'ecdysone au cours du cycle d'intermue chez le crabe Carcinus moenas; comparaison entre individus sains et parasités par Sacculina carcini

Radioimmunoassay is performed for a quantitative study of ecdysone in Carcinus moenas. Two periods can be determined in the moulting cycle. In haemolymph from ecdysis until beginning D0 stage, the moulting hormone content is not very high; from D0 stage until D4 stage, the hormone titer rises considerably. We can observe a similar rise in hormone content in both haemolymph and Y organ during the premolt stages. In sacculinized Crabs, the determined values of ecdysone in haemolymph and in Y organ are lower than those determined in healthy animals at the same stage.

[Protein composition of hemolymph from the crabs Carcinus mediterraneus Czerniavsky, healthy or parasitized by Sacculina carcini Thompson]. / Composition protéique de l'hémolymphe des crabes Carcinus mediterraneus Czerniavsky, sains ou parasités par Sacculina carcini Thompson

The proteic composition of haemolymph from C. mediterraneus varies during the intermoult cycle. Some proteic fractions are occasionally absent in post-ecdysis. At each intermoult stage, parasitized Crabs have generally the same protein pattern as healthy ones, but the presence of the observed fractions is not constant. Compared healthy Crabs, the sacculinized animals show a surnumerary proteic fraction. With very short delay, the injection of ecdysterone puts the proteic composition of haemolymph from sacculinized Crabs in order, this composition becomes comparable to that from healthy individuals.

[Biological methylations in the crab Carcinus maenas; in vitro inhibition by a fraction purified from androgen gland extracts]. / Méthylations biologiques chez le crabe Carcinus maenas; inhibition in vitro par une fraction purifiée extraite des glandes androgènes

Extracts from muscles, testis, seminal vesicles and ovaries of the Crab, Carcinus maenas, have been studied in vitro, in presence of [14C]-methyl S-adenosylmethionine, with an E. coli tRNA as methyl acceptor. The highest level of methylases is found in the testis. It has been reported previously that a purified fraction extracted from the androgenic glands of Carcinus maenas inhibits the vitellogenesis in ovaries. We now show that the same fraction inhibits tRNA methylation in an extract of testis as methylase; a 50% inhibition is obtained with about 10 mug of a purified fraction corresponding to 15 glands. With an enzymatic preparation from the ovaries, a 50% inhibition of the tRNA methylase is observed with the purified extract from 4 glands.

[Preliminary results on the ultrastructure of the molting gland (Y organ) of normal and Sacculina carcini parasitized crabs]. / Données préliminaires sur l'ultrastructure de la glande de mue (organe Y) chez le Crabe Carcinus mediterraneus sain en parasité par Sacculina carcini

The Y organ of Carcinus mediterraneus parasitized by Sacculina carcini and of normal Crabs at the C4 stage of the intermolt cycle have the same ultrastructure. At this stage the secretory activity of the Y organ appears to be reduced.