Relationship of body fat and body mass index in young Pacific Islanders: a cross-sectional study in European, Melanesian and Polynesian groups.

BACKGROUND: Body mass index is the most often used indicator of obesity but does not distinguish between lean and fat mass. Adiposity at the same body mass index differs across ethnic groups. OBJECTIVES: The twofold aim of this study was to determine whether body mass index (BMI)-based references are correlated with body fat percentage (%BF) in a pluri-ethnic population of Pacific Islanders and to assess the diagnostic accuracy of these references by using the percentage of body fat as the gold standard. METHODS: Height and weight were obtained, and triceps and subscapular skinfold thicknesses were measured in a sample of 796 adolescents (11-16 years) from the three main ethnic groups in New Caledonia: Melanesian, European and Polynesian. %BF was derived from the Slaughter equations, and BMI z score was calculated by using various international and national references. RESULTS: Melanesian teens had lower %BF compared with their European counterparts for the same BMI z score. Whatever the BMI-based reference used to detect overfatness (%BF >25% for boys and >30% for girls), sensitivity was higher in Melanesian adolescents, while specificity was higher in their European counterparts. Diagnostic accuracy was better in Melanesian compared with European adolescents. CONCLUSIONS: This study shows that Melanesian adolescents have lower %BF than their European counterparts for the same BMI z score. Therefore, the diagnostic accuracy of BMI to detect overfatness is related to ethnicity. Whatever the BMI-based reference, sensitivity was higher in the Melanesian group, while specificity was higher in the European group.

The effect of dietary protein restriction on the secretion of LH and FSH in pre-pubertal female lambs.

The effect of restricted dietary protein on the synthesis, storage and release of LH and FSH was studied in pre-pubertal female lambs. The experiment started when the lambs were aged 12 weeks and weighed 26.0+/-1.6 kg. It was conducted for 25 weeks. The lambs were fed isocaloric diets containing either a restricted level of crude protein (8% CP; n=6; treatment R) or an elevated one (18% CP; n=4; treatment E). At 37 weeks of age and before the first oestrous cycle, blood samples were collected over 6 h at 10 min intervals for LH assay. The lambs were slaughtered and their brains recovered and fixed in situ. Immuno-reactive (IR) LH and FSH cells were localised by immunohistochemistry techniques. Messenger RNA analyses used by non-isotope in situ hybridisation with sense and anti-sense riboprobes from beta subunits of LH and FSH cDNA clones. Data were generated using computer analysis to measure the proportion of IR and/or hybridising cells and their optical density for immuno-staining and hybridisation signal. Plasma LH was measured by RIA. The daily live-weight gains were 56.5+/-13.1 g and 97.8+/-14.3 g for R and E lambs, respectively (P<0.05), so that final weights at slaughter were 36.1+/-1.97 kg and 39.1+/-3.44 kg, respectively (P<0.05). The number of cells expressing LH beta mRNA and the optical density of this hybridisation signal was significantly (P<0.001) lower in the R lambs but the number of IR LH positive cells was higher (P<0.001) than for the E lambs. The concentration of LH in the plasma of R sheep was lower (P<0.05) than the E group and this response was associated with a decrease (P<0.05) in LH pulse frequency and amplitude. Dietary protein concentration appeared to have no effect on the IR in FSH cells or on the expression of FSH beta mRNA. In summary, the low protein diet influenced the body weight and weight gain of growing lambs and exerted an inhibitory effect on the synthesis and the release of LH in the pituitary gonadotrophs. No such effect was observed for FSH. It was concluded that the protein concentration of the diet consumed during the growth of female lambs may be an important modulator of processes leading to the pre-pubertal rise in LH.

Modulation of luteinizing hormone subunit gene expression by intracerebroventricular microinjection of gonadotropin-releasing hormone or beta-endorphin in female rats.

The effects of gonadotropin-releasing hormone (GnRH), beta-endorphin and its antagonist naloxone on the expression of luteinizing hormone (LH) subunit genes and LH secretion were examined in ovariectomized and/or cycling female rats through their direct microinjection into the third cerebral ventricle, in the proximity of the hypothalamus-pituitary complex. GnRH (1 nM) induced a significant augmentation of the pituitary content of alpha mRNA when administered 15, 30 or 60 min intervals over 5 h to ovariectomized rats whereas only the 30 and 60 min intervals were effective in increasing LHbeta mRNA, and the 60 min intervals for LH release. This was in agreement with the established concept of a pulse-dependent regulation of gonadotropin synthesis and release. Hourly pulses of GnRH also increased alpha and LHbeta mRNA levels when microinjected in female cycling rats during proestrus or diestrus II. Using this model we observed a marked negative influence of hourly intracerebral microinjections of beta-endorphin on LH mRNA content and LH release in ovariectomized rats while naloxone had no effect. This suggests that endogenous beta-endorphin was unable to exert its negative action on beta-endorphin receptors that were present and responded to the ligand. The present approach would be valuable for the exploration of the mechanisms of action of beta-endorphin or other substances on the functions of the gonadotrophs.

Functional importance of transmembrane helix 6 Trp(279) and exoloop 3 Val(299) of rat gonadotropin-releasing hormone receptor.

Previous studies have established that the interaction of gonadotropin-releasing hormone (GnRH) with its receptor (GnRHR) would require partial entry of the N- and C-terminal regions of ligand into the transmembrane core. The functional significance of the conserved aromatic residue Trp(279) present in the transmembrane helix 6, and Val(299) located in exoloop 3 of the rat GnRHR was investigated by mutagenesis followed by expression in Chinese hamster ovary-K1 cells. Compared with wild-type, substitution of Trp(279) with Ser or Arg resulted in a marked reduction or total abolition, respectively, of ligand binding and, in both cases, abrogation of GnRH-induced inositol phosphate production. A total absence of functionality was observed when Val(299) was simply replaced with Ala. Mention should be made that an expression of all mutated and wild-type receptor proteins was observed. Interestingly, the double mutant [Trp(279)Arg/Val(299)Ala]GnRHR restored B(max) to wild type (504 +/- 43 versus 541 +/- 41 fmol/mg protein), but with a diminished affinity (4.95 +/- 1.05 versus 0.94 +/- 0.35 nM), and GnRH failed to induce inositol phosphate. No influence of the mutations was seen on internalization of the receptor. The three-dimensional model of GnRH binding to the rat GnRHR was built predicting that Trp(279) is buried at 20 A in the transmembrane core of the receptor, directly in contact with Trp(3) of GnRH. In contrast, Val(299) is located in a region that cannot be precisely defined at the extracellular end of transmembrane helix 7. Although models cannot provide any clue concerning the observed interactivity between the two distal residues, altogether these data reveal the functional importance of both GnRHR Trp(279) and Val(299) and suggest that Trp(279), interacting with GnRH Trp(3), represents the bottom of the binding pocket.

LH down-regulates gonadotropin-releasing hormone (GnRH) receptor, but not GnRH, mRNA levels in the rat testis.

The demonstration of an inhibitory effect of gonadotropin-releasing hormone (GnRH) agonists upon steroidogenesis in hypophysectomized rats and the presence of mRNA coding for GnRH and GnRH receptors (GnRH-R) in rat gonads suggests that GnRH can act locally in the gonads. To assess this hypothesis, we investigated the effects of GnRH analogs, gonadotropins and testosterone on the levels of both GnRH and GnRH-R mRNA in the rat testis. Using dot blot hybridization, we measured the mRNA levels 2 to 120 h after the administration of the GnRH agonist, triptorelin. We observed an acute reduction of both GnRH and GnRH-R mRNAs 24 h after the injection (about 38% of control). However, the kinetics for testis GnRH-R mRNA were different from those previously found for pituitary GnRH-R mRNA under the same conditions. Initially, the concentrations of serum LH and FSH peaked, then declined, probably due to the desensitization of the gonadotrope cells. In contrast, the GnRH antagonist, antarelix, after 8 h induced a 2.5-fold increase in GnRH-R mRNA, but not in GnRH mRNA, while gonadotropins levels were reduced. Human recombinant FSH had no significant effect on either GnRH or GnRH-R mRNA levels. Inversely, GnRH-R mRNA levels markedly decreased by 21% of that of control 24 h after hCG injection. Finally, 24 h after testosterone injection, a significant increase in GnRH-R mRNA levels (2.3 fold vs control) was found, but a reduction in the concentration of serum LH, probably by negative feedback on the pituitary, was observed. In contrast, GnRH mRNA levels were not significantly altered following testosterone treatment. Since LH receptors, GnRH-R and testosterone synthesis are colocalized in Leydig cells, our data suggest that LH could inhibit the GnRH-R gene expression or decrease the GnRH-R mRNA stability in the testis. However, this does not exclude the possibility that GnRH analogs could also affect the GnRH-R mRNA levels via direct binding to testicular GnRH-R. In contrast, the regulation of GnRH mRNA levels appeared to be independent of gonadotropins. Taken together, our results suggest a regulation of GnRH and GnRH-R mRNA specific for the testis.

GnRH-dependent up-regulation of nitric oxide synthase I level in pituitary gonadotrophs mediates cGMP elevation during rat proestrus.

We have recently provided evidence that the concentration and activity of the enzyme nitric oxide synthase type I (NOS I) was stimulated in gonadotrophs by GnRH, suggesting a role for the NOS I/NO pathway in the GnRH control of cell functions. To further establish the GnRH regulation of pituitary NOS I under physiological circumstances, we have examined the expression of NOS I during the 4-day estrus cycle in rats. Western blot analysis demonstrated that NOS I was present in the anterior pituitary at low levels during diestrus I (DI) and diestrus II, then was subject to a significant increase on the afternoon of proestrus to reach a maximal 3-fold increase between 19:00 and 20:00 h, after which NOS I decreased to return, during estrus, to levels within the range detected in diestrus. No such variation was apparent in the posterior pituitary lobe. NADPH-diaphorase histochemistry combined with immuno-identification of the cells revealed that active NOS I was expressed in both the gonadotrophs and the folliculo-stellate cells throughout the whole cycle but only the gonadotrophs showed an up-regulation during proestrus. A high temporal correlation was observed in the profiles of NOS I, pituitary cGMP and serum luteinizing hormone (LH) suggesting an implication of GnRH. Consistently, the administration of a potent GnRH antagonist to rats totally abolished the rise in pituitary NOS I and cGMP, in addition to suppressing, as expected, the surge in the secretion of LH. A role of NOS I as a mediator in the GnRH-induced augmentation in cGMP was further established in vitro by incubating anterior pituitaries in the presence of the NOS inhibitor, L-NMMA (1 mM). Altogether, these data demonstrate that the level and activity of NOS I is up-regulated in gonadotrophs during proestrus, in a manner consistent with a major implication of GnRH over a period during which its release from the hypothalamus, as well as gonadotroph responsiveness, are at maximum. This effect is accompanied by a NOS/NO-mediated rise in cGMP. In the absence of obvious effect on gonadotropin release, the roles of NO and cGMP in the regulation of gonadotroph functions, especially during proestrus, remain to be established.

Evidence that gonadotropin-releasing hormone stimulates gene expression and levels of active nitric oxide synthase type I in pituitary gonadotrophs, a process altered by desensitization and, indirectly, by gonadal steroids.

To determine the site and mechanism of action of gonadal steroids on pituitary nitric oxide synthase type I (NOS I), present in both gonadotrophs and folliculo-stellate cells, the effects of castration and steroids were examined in male rats, in the presence of a GnRH antagonist (Antarelix). Western analysis showed a rapid and substantial increase with time, after orchidectomy, of NOS I protein, the concentration doubling in 24 h and reaching a maximal 4- to 5-fold increase after 3-7 days, followed by a progressive decline after 2 weeks. Testosterone or estradiol replacement, or administration of GnRH antagonist, totally abolished the effects of castration, demonstrating a mediation of the steroid effects via GnRH. In noncastrated rats, steroids and the GnRH antagonist also caused a reduction in the levels of NOS I (by 50-60%), consistent with inhibition of endogenous GnRH stimulation. In marked contrast, administration of a potent GnRH agonist (Triptorelin) to intact rats increased the levels of NOS I. A time-course study with a long-lasting formulation showed that rise in NOS I developed rapidly after a lag of approximately 5 h, with a 2-fold increase detectable after 8 h and a maximal 4.5-fold after 48 h. The level declined afterwards in a manner consistent with homologous desensitization that may occur in the continuous presence of GnRH; however, the profile was different and delayed compared with those of gonadotropin release. As observed for NOS I protein, NOS I messenger RNA concentration was increased by castration or GnRH agonist and reduced by steroids or GnRH antagonist. Taken together, these data demonstrate that steroids indirectly regulate NOS I messenger RNA and protein levels, through the hypothalamic modulation of GnRH, which represents the primary regulator of NOS I. No effect of steroids on NOS I was seen in the posterior lobe. NADPH-diaphorase histochemistry coupled to immuno-identification of the cells revealed that the treatments affecting the concentration of NOS I concomitantly altered the activity but exclusively in gonadotrophs and not in folliculo-stellate cells (which do not respond to GnRH), reinforcing the idea that GnRH played a major regulatory role. Expression in gonadotrophs of a GnRH-dependent NOS I and the ensuing production of nitric oxide represents a potentially novel signaling pathway for the neuropeptide in the anterior pituitary, consistent with the previously reported GnRH-induced cGMP production, the role of which remains to be evaluated.

Testosterone inhibits the basal and gonadotropin-releasing hormone-stimulated synthesis and release of newly synthesized alpha- and lutropin (LH) beta-subunit but not release of stored LH in cultured rat pituitary cells.

To further explore the mechanism of steroid feedback in male, the effects of testosterone (T) and gonadotropin-releasing hormone (GnRH) on the rates of alpha- and lutropin (LH)beta-chain synthesis, neosynthesized subunits and radioimmunoassayable LH release into the medium were studied in the cultures of anterior pituitary cells from orchiectomized and intact rats. Polypeptides were [35S]methionine-labeled, immunoprecipitated separately in the medium and cells, then after SDS-PAGE precisely quantified. The total (medium + cells) radioactivity incorporated in the absence of GnRH into alpha- and LH beta-subunit was increased in orchiectomized rat cells vs. intact rat cells. GnRH stimulated the synthesis of both subunits, whether cells were from normal or castrated rat. T suppressed basal and GnRH-enhanced synthesis of both subunits in castrated rat cells. The values became closed to those observed in the normal rat cells. Also release of neosynthesized subunits from castrated rat cells into the culture medium was inhibited by T. In contrast, T did not change the basal and GnRH-induced radioimmunoassayed LH release. These results show that T can inhibit directly, at the pituitary level, alpha- and LH beta-subunit synthesis and neosynthesized but not stored LH release. They could explain, at least in part, no correlation between modifications of GnRH and LH secretion observed in vivo in response to T replacement.

Expression of gonadotropin-releasing hormone (GnRH) receptor gene is altered by GnRH agonist desensitization in a manner similar to that of gonadotropin beta-subunit genes in normal and castrated rat pituitary.

It was previously established that the administration of a potent GnRH agonist such as triptorelin (D-Trp6-GnRH) induced desensitization of pituitary gonadotropic cells, resulting in decreased expression of gonadotropin beta-subunit genes and the suppression of LH and FSH synthesis and release. Binding of GnRH to the pituitary is also affected by agonist treatment. To examine the desensitizing effects of GnRH agonist on the expression of the pituitary GnRH receptor (GnRH-R) gene, male rats were given triptorelin (long-acting formulation, 300 micrograms/kg), and levels of GnRH-R messenger RNA (mRNA) were determined by Northern and dot blot hybridization to a 32P-labeled rat complementary DNA probe. Abundances of gonadotropin alpha-subunit, LH beta, and FSH beta mRNAs were examined in parallel, using appropriate probes. A rapid time-dependent decrease in the level of GnRH-R mRNA was observed in rats after triptorelin administration. A minimum residual level of mRNA, in the range of 20-25% of the initial value, was attained as early as 5 h after treatment. Levels further stabilized to 25-30% after a small transient increase to 45% on day 5. A single injection was effective for at least 30 days, after which GnRH-R mRNA levels slowly returned to normal, suggesting a progressive abolition of agonist effects. A concomitant acute depletion of mRNA levels was observed for LH beta and FSH beta (50% decrease in about 48 and 3 h, respectively), whereas the alpha-subunit message increased (rapidly reaching a level 1.8-fold that in control rats after 1-2 days). Castration induced a 3.8-fold elevation in the amounts of GnRH-R mRNA after 3 weeks, whereas alpha, LH beta, and FSH beta mRNAs increased by 6.2-, 7.9-, and 4.2-fold, respectively, compared to corresponding values in intact animals. Administration of the GnRH agonist readily prevented, for as long as 3 weeks, the stimulatory effects of castration on the GnRH-R mRNA and mRNAs for the beta-subunit of gonadotropins, but not for the alpha mRNA, which remained at a high level. When triptorelin was administered 3 weeks postoperatively, the castration-induced increase in LH beta and FSH beta was totally abolished, and no significant effect was noted on alpha-subunit mRNA. In conclusion, these data demonstrate that expression of the GnRH-R gene is subject to regulation and depends on GnRH stimulation, in a manner that indicates susceptibility to desensitizing action by the long-acting GnRH analog, triptorelin.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of somatostatin receptors and growth inhibition by the somatostatin analogue BIM23014 in small cell lung carcinoma xenograft: SCLC-6.

Human small cell lung cancer (SCLC) is a neuroendocrine tumour with a very poor prognostic. Receptors for somatostatin-14 and its synthetic analogue BIM23014 (Lanreotide) were characterized in 3 human SCLC xenografts (SCLC-6, SCLC-10 and SCLC-75) transplanted in nude mice. The binding activity of both iodinated ligands was tested by cross-linking assay. One major complex of 57kDa was identified by both ligands in all 3 tumours. Two other minor complexes were only detected by the natural ligand: 90kDa in all 3 tumours and 70kDa in 2 out of the 3 tumours (SCLC-6 and SCLC-75). Analysed by Northern hybridization, the expression of the gene encoding for the receptor subtype I was detected in all 3 tumours whereas the expression of the receptor subtype II was only detected in 2 out of the 3 tumours (SCLC-6 and SCLC-75). No receptor subtype III transcript was observed. The relative quantification of the detected messengers and of the cross-linked complexes determined by densitometry suggested that SCLC-6 contained a large amount of somatostatin receptors. SCLC-6 growing in nude mice was used to evaluate the antiproliferative effect of BIM23014. BIM23014 (250 micrograms, b.i.d. for 5 days) significantly inhibited tumor growth and had an additive effect with cis-platinum (1.5 mg/kg/day for 2 days) when given concomitantly. Values of the relative tumour volume as compared to control were: BIM23014 alone 57%, cis-platinum alone 57% and BIM23014 + cis-platinum: 78%. These experimental data suggest that BIM23014 given alone or in combination with cis-platinum could have a therapeutic potential in the treatment of somatostatin receptor positive SCLCs.

[Recent data on the gonadoliberin receptor and the neuropeptide control mechanisms for gene expression of gonadotropin hormones]. / Données récentes sur le récepteur de la gonadolibérine et les mécanismes de contrôle par le neuropeptide de l'expression des génes des hormones gonadotropes.

GNRH plays a pivotal role in the neurohormonal control of reproduction by promoting hte secretion of pituitary gonadotrophins, LH and FSH. GnRH also stimulates the synthesis of constitutive gonadotrophin subunits alpha and beta and its own receptor number. Gonadotrophin synthesis appears to be regulated by GnRH through various molecular mechanisms that include, in a complementary and in some cases differential manner, enhanced transcriptional activity of subunit genes and polyadenylation of transcripts. The latter is known to result in increased stability and/or translational activity of mRNAs. These effects of GnRH are mimicked by the direct activation of protein kinases A and C, two different but possibly interconnected signalling pathways that may account for the pleiotropic and concerted alterations of both synthesis and release of gonadotrophins. GnRH operates on the gonadotropic cell level via a transmembrane, G-protein coupled receptor, the structure of which has recently been determined by molecular cloning. This receptor differs from the other members of hte super-family essentially by a rather short length (only 327-328 amino acids) and a truncated carboxyterminus. Recent experiments suggest a genomic control of the GnRH receptor synthesis, especially by GnRH itself, the importance, and role of which remains to be established for the pituitary gonadotropic function.

Messenger ribonucleic acids for alpha- and beta-isoforms of cyclic adenosine 3',5'-monophosphate-dependent protein kinase subunits present in the anterior pituitary: regulation of RII beta and C alpha gene expression by the cyclic nucleotide and phorbol ester.

Expression of the genes encoding the alpha- and beta-isoforms of the catalytic (C) and regulatory type I (RI) and type II (RII) subunits of cAMP-dependent protein kinases (PKA) in the anterior pituitary gland of rats was examined by hybridization of specific 32P-labeled cDNA probes to mRNA. C alpha, C beta, RI alpha, RI beta, RII alpha, and RII beta mRNAs were all present in the anterior pituitary gland and cultured cells, but in different proportions; C alpha was the most abundant, and RII alpha was the least. For C alpha, C beta, RI beta, and RII alpha, Northern blot analysis revealed single mRNAs with sizes of 2.4, 4.5, 2.8, and 6.0 kilobases (kb), respectively. For RI alpha, three distinct mRNAs (1.7, 3.2, and 3.7 kb) were identified, and for RII beta, two were found (1.8 and 3.5 kb). Compared to other tissues and species, including ovine and rat pituitary and testis, differences in sizes and relative abundance of mRNAs and/or mRNA subtypes were observed, demonstrating that great diversity exists, which may reflect tissue and species variation in posttranscriptional processing of mRNA transcripts. When rat anterior pituitary cells were cultured in the presence of 8-bromo-cAMP (8-Br-cAMP), RII beta mRNA levels increased in a dose- and time-dependent manner to a maximum of about 4- to 5-fold the basal values after 5 h in the presence of 2 mM 8-Br-cAMP. Under the same conditions, the C alpha mRNA, already at high levels, further increased, but to a lesser extent (1.5-2 times compared to basal abundancy). Cholera toxin (6 nM) reproduced the effects of 8-Br-cAMP on both C alpha and RII beta mRNAs. These effects were completely abolished in the presence of actinomycin-D, suggesting that cAMP enhances RII beta and C alpha gene transcription. On the other hand, cell exposure to the phorbol ester 12-O-tetradecanoyl 13-phorbol acetate, a potent activator of protein kinase-C (PKC), readily induced a dose-dependent actinomycin-D-inhibited increase in the RII beta mRNA content to levels about 2-fold those in control unstimulated cells, whereas it depressed levels of C alpha mRNA by 25%. In conclusion, we have found that the six genes coding for the alpha- and beta-isoforms of PKA subunits are all expressed in the anterior pituitary, and that the expression of two of those genes (RII beta and C alpha) is regulated by cAMP and phorbol ester in a similar or opposite manner.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential stability of mRNAs coding for alpha and gonadotropin beta subunits in cultured rat pituitary cells.

Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) result from the assembly of a common subunit alpha and a unique subunit beta, expressed in the same cell by single, structurally-related genes. In order to compare the intrinsic stability of the alpha, LH beta and FSH beta mRNA transcripts, we used cultured rat pituitary cells incubated in presence of actinomycin D. Hybridization with 32P-labelled rat cDNA probes showed that the cell content of all three mRNAs decreased with time, but at different rates. Apparent half-lives, estimated as the time necessary to observe a 50% mRNA decay, were 1.0 +/- 0.13 h for FSH beta, 6.5 +/- 0.25 h for alpha and 44 +/- 0.5 h for LH beta, stability thus exhibiting an inverse relation to the sizes of the corresponding mRNAs (approximately 1700, 800 and 700 nucleotides, respectively). Northern analysis revealed that the decline in mRNA abundance was associated with a progressive decrease in the length of mRNAs, most clearly visible for alpha and LH beta. For the most stable LH beta mRNA, shortening was apparent as early as 2 h after exposure to actinomycin D thus preceding neatly the decrease in amount starting at about 10-12 h. In vitro RNase H digestion demonstrated that shortening resulted from a reduction of the length of the poly(A) tract. These data establish that the three mRNAs coding for gonadotropin subunits have different stabilities although they share substantial homology. Diversity in size and sequence essentially resides in untranslated regions in which, we suggest, specific motifs and protein factors may interact to determine mRNA stability.(ABSTRACT TRUNCATED AT 250 WORDS)

[Focus on the biogenesis of hypophyseal gonadotropins]. / Mise au point sur la biogénèse des gonadotropines hypophysaires.

We have studied the regulation of the biosynthesis of pituitary gonadotropins in the rat by gonadal steroids and a hypothalamic hormone, GnRH. The methodology used for studying the action of steroids, was either cell-free translation of pituitary messenger RNAs, or hybridization (Northern blot) with synthetic oligodeoxynucleotides (ODN), and for studying the effect of GnRH, primary anterior pituitary cell culture. Our results show that gonadectomy increases and injection of gonadal steroids into gonadectomized rats diminishes the rate of synthesis of the gonadotropin subunit precursors. Progesterone acts only after induction of its pituitary receptors in ovariectomized rats with estradiol benzoate. Thyroxine modulates the action of steroids. Hybridization experiments suggest that gonadal steroids act on the expression of genes encoding the precursors of gonadotropin subunits. GnRH significantly increases incorporation of the labeled amino acids into polypeptide chains of both alpha and LH beta subunits. Intracellular mediators of hormone action, such as cyclic AMP and diacylglycerols, mimic the stimulatory action of GnRH on the synthesis of LH subunits. However, we have no evidence that these products intervene in the effect of GnRH on the LH subunit synthesis. In conclusion, the synthesis of LH and FSH subunits is regulated, with opposite effects, by gonadal steroids which exert their negative control at the genomic level and by GnRH which proceeds via different, yet unknown mechanisms.

[Stimulation of LH gene expression by GnRH. Role of protein kinases A and C]. / Stimulation de l'expression des gènes de LH par le GnRH. Rôle des protéines kinases A et C.

Using anterior pituitary cells in culture, incubated in the presence of [35S] Met, we have previously demonstrated that GnRH stimulates the synthesis of LH polypeptide chains, an effect which was reproduced in a non additive manner by direct activation of protein kinases A and C (by cyclic AMP or diacylglycerol analogues, respectively), and abolished by actinomycin D. The respective roles of GnRH and protein kinases A and C in the stimulation of LH subunit gene expression was examined in the present study by evaluating the effects on the cellular levels of alpha and LH beta mRNAs of optimal concentrations of Buserelin (0.1 nM) an undegradable GnRH analogue, cholera toxin (6 nM) a cyclic AMP (AMPc) generator and 12-O-tetradecanoyl phorbol 13-acetate (TPA) a diacylglycerol analogue. The specific mRNAs were quantified by densitometry analysis of Northern blot hybridization using 32P-labeled single strand cDNAs. We observed TPA, like Buserelin, increased LH mRNA levels in a very similar manner, the alpha mRNA increasing 1.8 - 2.5 fold and the LH beta mRNA 1.4 - 1.7 fold after 5 h culture and 3.0 - 3.5 fold and 2.2 - 2.4 fold respectively, after 24 h. In the presence of cholera toxin, the changes were more rapid, the highest values being reached at 5 h (8.6 fold increase for alpha and 4.0 fold for LH beta mRNA). LH, radioimmunoassayed in parallel in the cell medium, increased 5.9 fold after 5 h and 7.1 fold after 24 h culture in the presence of Buserelin, and 2.9 fold and 5.4 fold, respectively, in the presence of TPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic AMP enhances gene expression, synthesis and release of newly synthesized alpha and luteinizing hormone beta subunits in cultured rat anterior pituitary cells.

We have previously demonstrated that GnRH stimulates the synthesis of both the ? and LH? polypeptide chains, an effect which was reproduced in a non additive manner by direct activation of protein kinases A and C, and abolished by actinomycin D. In the present study, we examined the effects in monolayer cultures from rat anterior pituitary cells of 8-Br-cAMP and cholera toxin, on ? and LH? subunit mRNA levels and in parallel the synthesis and release of the subunits. RNA blot hybridization analysis with cDNA probes demonstrated that ? and LH? mRNA levels increased by 8.9- and 4.7-fold, respectively, after a 5 h-incubation in the presence of 6 nM cholera toxin and 7.1- and 2.9-fold in the presence of 1.5 mM 8-Br-cAMP. Under the same conditions, [(35)S]methionine incorporation into ? and LH? subunits was optimally stimulated, by 2.8-fold and 1.7 to 2.2-fold, respectively, whether the cAMP analogue 8-Br-cAMP or cholera toxin, an endogenous cAMP generator, were employed. Further, in addition to synthesis, 8-Br-cAMP appeared to increase release of neosynthesized ? and LH? polypeptides, and in this respect, 8-Br-cAMP was more effective than GnRH. In contrast, 8-Br-cAMP had a weak, non significant effect compared to GnRH on the release of total radioimmunoassayable LH in the cell media. These data provide the first direct evidence for a stimulation of ? and LH? gene expression by cyclic AMP, which accounts for an increase in subunit synthesis. They suggest that cAMP, as previously shown for diacylglycerols, is a potent candidate for an intracellular mediator of the GnRH effects on subunit synthesis and that it is largely responsible for sustained (protein synthesis-dependent) LH release.

[Ambivalent effect of thyroxine on the expression of gonadotropin genes in normal and orchidectomized rats]. / Effet ambivalent de la thyroxine sur l'expression des gènes des gonadotropines chez le rat intact et gonadectomisé.

In order to examine the role of thyroid hormones on pituitary gonadotropin synthesis and release, normal, thyroidectomized and orchidectomized male rats were treated in parallel by daily injections of thyroxine (10 micrograms/10 g). Pituitary levels of alpha, LH beta, FSH beta and TSH beta mRNA were determined using two complementary approaches: 1) cell-free translation of pituitary mRNA and quantitative immunoprecipitation of labeled subunit precursors, and 2) hybridization with [32P]-labeled cDNA probes. In intact and thyroidectomized rats, injection of thyroxine (T4) resulted in the depletion of all mRNAs encoding the glycoprotein hormone subunits. In contrast, when rats received T4 three weeks after orchidectomy, levels of alpha, LH beta- and FSH beta-mRNA, but not TSH beta-mRNA increased, by 2, 1.5 and 1.2 times, respectively, compared to castrated, sham-injected rats. LH was assayed in serum and pituitary. In intact rats, T4 decreased pituitary content and release of LH by about 25%. In castrated rats, T4 antagonized the gonadectomy-induced increase in serum LH and increased the pituitary content in LH to levels consistent with an increased synthesis. In conclusion, T4 treatment increases or decreases levels of translatable mRNA encoding all 3 gonadotropin subunits depending on whether rats were castrated or not, respectively. Thus, T4 appears to exert a modulatory role in the regulation of gonadotropin gene expression by steroids. This effect of T4 on the synthesis is discordant with the effects of T4 on the release of LH and probably involves complex, multimodal control mechanisms.

Rat placental mRNA directs the synthesis of a polypeptide immunologically related to alpha-subunit of glycoprotein hormones.

In order to investigate the existence of a chorionic gonadotropin (CG) in the rat, placental mRNA was prepared from either the foetal disc or the maternal site of implantation in pregnant rats and translated in a wheat-germ cell-free translation system in the presence of 35S-labeled methionine and cysteine. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the radioactive material immunoprecipitated using antiserum specific to native rat alpha-subunit allowed us to isolate in translation products from both sources (foetal disc and maternal site of implantation) a single polypeptide of 17 kDa having the electrophoretic properties of the rat pituitary alpha-precursor. This polypeptide was absent in media derived from translation of mRNA extracted from uterine horn of non-pregnant female rats, and was approximately 10 times more abundant when RNA was derived from the maternal part (about 0.25% of cpm in total proteins) rather than the foetal part (0.026%) of the placenta. Immunoprecipitation was prevented in the presence of an excess of rat (but not ovine) alpha-subunit. Antisera directed against denatured bovine alpha-subunit, which cross-react with the rat pituitary alpha-precursor, did not bind the placental peptide. These results suggest that this rat placental polypeptide and the rat alpha-subunit of pituitary glycoprotein hormones have important differences in their primary structure, but share discrete structurally and/or conformationally related regions in their polypeptide chains. The possibility that these partial homologies account for gonadotropin-like activity of a presumed rat CG remains to be ascertained.

[Hormonal regulation of the expression of genes coding for pituitary gonadotropins]. / Régulation hormonale de l'expression des gènes codant pour les gonadotropines hypophysaires.

In the rat, gonadectomy increases and injection of estradiol or of testosterone depresses the translational capacity of mRNAs encoding gonadotropin subunits alpha, LH beta and FSH beta. This inhibitory effect of steroids can also be demonstrated in vitro using pituitary cells in culture. Thus, steroids appear to regulate gonadotropin synthesis, at least in part, by a direct action on the pituitary gland. Hybridization experiments, using labeled cDNA probes, show gonadal steroids to act by decreasing the number of copies of specific mRNAs encoding each one of the gonadotropin subunits. Definite evidence has been obtained that GnRH stimulates biosynthesis of the polypeptide chains of LH subunits, thus confirming our previous observations. Preliminary results suggest cAMP could be involved in this process as a mediator of GnRH action. Although actinomycin D prevents the stimulatory effect of GnRH on gonadotropin biosynthesis, the translational capacity of specific mRNAs encoding LH subunits did not increase after incubation of the cells with GnRH, thus suggesting that GnRH does not act directly either on the gonadotropin subunit genes or on the intrinsic messenger activity of mRNAs encoding the gonadotropin subunit precursors. Our data demonstrate that the synthesis of pituitary gonadotropins is under a dual hormonal control, by gonadal steroids and GnRH. We suggest that the negative control by steroids and stimulatory effect of GnRH occur via different routes, the former at the genomic level, the latter at another step which remains to be elucidated.