Yannick Lerrant

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...

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.

Differential stability of mRNAs coding for α and gonadotropin β subunits in cultured rat pituitary cells

Abstract Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) result from the assembly of a common subunit α and a unique subunit β, expressed in the same cell by single, structurally-related genes. In order to compare the intrinsic stability of the α, LHβ and FSHβ 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 FSHβ, 6.5± 0.25 h for α and 44 ± 0.5 h for LHβ stability thus exhibiting an inverse relation to the sizes of the corresponding mRNAs (~ 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 α and LHβ. For the most stable LHβ 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. The presence and length of the poly(A) stretch would also play a role.

Intracerebroventricular infusion of neuropeptide Y up-regulates synthesis and accumulation of luteinizing hormone but not follicle stimulating hormone in the pituitary cells of prepubertal female lambs

Neuropeptide Y (NPY) is a putative neuroregulator of the reproductive axis in the central nervous system. In this study we evaluated the effects of central infusion of exogenous NPY on the secretory activity of pituitary gonadotrophic cells in prepubertal lambs. Immature female Merino sheep (n=12) were infused of Ringer solution (control) or 50 microg of NPY to the third ventricle for 5 min and then slaughtered 3 h later. Immunoreactive luteinizing hormone (LH) and follicle stimulating hormone (FSH) cells were localised by immunohistochemistry using antibody raised against LHbeta and FSHbeta. Messenger RNA analyses were performed by in situ hybridisation using sense and antisense riboprobes produced from beta subunits of LH and FSH cDNA clones. The results were generated by computer image analysis to determine the area fraction occupied by immunoreactive and/or hybridising cells and optical density for immunostaining and hybridisation signal. LH in the blood plasma was determined by radioimmunoassay. It was found, that in the lambs infused with NPY the area fraction and optical density for immunoreactive LH cells and mRNA LHbeta-expressing cells increased significantly (P<0.001), compared to the vehicle-infused animals. The concentration of LH in the blood plasma did not differ between control and treated groups. The NPY infusions had no effect on the immunoreactivity of FSH cells or on expression of mRNA for FSHbeta. In conclusion we suggest that NPY may be an important component of mechanisms stimulating the synthesis and storage but not the release of LH in the pituitary gonadotrophs from prepubertal female sheep. In addition, this effect is specific for LH, no such effect was apparent on FSH.

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.

Testosterone inhibits the basal and gonadotropin-releasing hormone-stimulated synthesis and release of newly synthesized α- and lutropin (LH) β-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.

In vivo modulation of follicle-stimulating hormone release and β subunit gene expression by activin A and the GnRH agonist buserelin in female rats

The effects of separate and simultaneous recombinant bovine (rb) activin A and buserelin administration on the FSH release and pituitary FSH beta subunit gene expression in vivo were examined in ovariectomised, estradiol pretreated rats. The animals received a single injection of either rb activin A (50 ng), buserelin (1 micro g) or activin/buserelin (50 ng+1 micro g/0.1 ml PBS) into the jugular vein and were killed 30 min, 1, 3 and 5h later. Activin A stimulated FSH release and effect appeared 1h after injection (168% increase of controls) reaching a maximum at 3h (437% of controls). Activin A and buserelin exerted their effects with a distinct time courses: activins stimulation was not so rapid when compared with buserelin. The simultaneous administration of rb activin A and buserelin amplified FSH release (118, 309, 1006 and 779% of controls). The low dose of activin A was sufficient to elevate FSH beta mRNA level as early as 3 and 5h after administration (170 and 140%, respectively). Activin plus buserelin stimulation resulted in a higher (340 and 360% of controls) FSH beta gene expression than after their separate administration. These results suggest that activin and buserelin may act independently and synergistically in the regulation of FSH release and beta subunit mRNA level.

Rat placental mRNA directs the synthesis of a polypeptide immunologically related to α-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.

Mise au point sur la biogenese 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.