Anna Starzec

Effect of gonadectomy on pituitary levels of mRNA encoding gonadotropin subunits and secretion of luteinizing hormone.

Using cell-free translation of pituitary mRNAs we have investigated how, following gonadectomy in rats, the translational capacity of the specific messages encoding precursors to gonadotropin subunits alpha, LH-beta and FSH-beta increases with time. In parallel, serum LH was assayed in order to compare release and synthesis patterns. We observed a rapid rise in the rate of synthesis of all three precursors, with a significant increase already detectable 4 days after gonadectomy, and a plateau reached after 21 days. The kinetics were similar in both males and females, but maximal translational values for alpha-subunit were slightly higher in males. During the same time period, serum LH rapidly increased in the males, while in the females the rise of circulating LH was somewhat delayed. Although no direct correlation seems to exist between synthesis and release processes of gonadotropins, it is evident from our previous findings and the present data that both phenomena are dependent on gonadal steroids. In this respect, estradiol has been shown to regulate negatively, via different routes, the synthesis as well as the secretion of pituitary gonadotropins.

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.

Detection of a lag phase in gonadotropin-releasing hormone stimulated synthesis of lutropin peptide chains in cultured rat anterior pituitary cells.

We have studied the time course (0-5h) of the stimulatory effect of the hypothalamic gonadotropin-releasing hormone (GnRH) on the biosynthesis of lutropin (LH) polypeptide chains, as measured by the incorporation of [35S] methionine into proteins synthesized in cultured rat anterior pituitary cells in the absence or presence of 10nM GnRH. Labeled polypeptides, immunologically related to LH subunits alpha and beta, were isolated by specific immunoprecipitation, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then revealed by fluorography and quantified by counting the excised bands. This methodology allowed us to detect the radioactivity incorporated into LH subunits after less than 15 min of incubation. During first 1h of the time-course the quantity of [35S]Met incorporated into both alpha and LH beta subunits was not increased by the presence of GnRH in the incubation medium. A significant increase in the incorporation of radioactivity into LH subunits was observed after 2h of GnRH treatment. However, the increase in LH release into the medium in response to GnRH, as measured by RIA, was immediate. These data demonstrate that GnRH-stimulated synthesis of LH polypeptide chains occurs after a lag of approximately 1h and involves mechanisms different from those governing the stimulation of LH release.

Effects of maternal deprivation on the adrenocorticotrophic and gonadotrophic axes in the hypothalamo–pituitary unit of preweanling female sheep: The histomorphometric approach

This study was designed to investigate the histochemical effects of maternal deprivation on the adrenocorticotrophic and gonadotrophic axes in the hypothalamo-pituitary unit of preweanling lambs. Twelve-week-old female lambs were divided into either the control (lambs reared under undisturbed maternal conditions; n=3) or the maternally deprived group (lambs separated for three days from their dams; n=3). The corticotrophin-releasing hormone (CRH) and gonadotrophin-releasing hormone (GnRH) in the median eminence and the adenohypophyseal adrenocorticotrophin (ACTH), gonadotrophins (LH and FSH) and mRNAs for their beta-subunits were investigated using the immunohistochemistry or hybridohistochemistry. In maternally deprived lambs, the percentage of the area occupied by immunoreactive (ir)-CRH nerve terminals was lower (P<0.05) and the percentage of the adenohypophyseal area (PAA) occupied by ir-ACTH cells was higher (P<0.05) compared with the control lambs. In the hypothalamo-gonadotrophic axis of maternally deprived lambs the percentage of area occupied by ir-GnRH nerve terminals was higher (P<0.05) and the PAA occupied by ir-FSHbeta cells was lower (P<0.05) in comparison with controls. The PAA occupied by gonadotrophs detected using hybridohistochemistry was higher (P<0.05) for LHbeta-mRNA in contrast to a lower (P<0.05) percentage for FSHbeta-mRNA in maternally deprived lambs compared with those staying with dams. In conclusion, maternal deprivation affected the accumulation of CRH and ACTH. The different and more striking alterations in FSH synthesis and storage in comparison with those concerning LH were observed in maternally deprived lambs. Thus, rupture of the preweanling young-mother social contact can affect the gonadotroph population activity, especially that relating to FSH-producing cells in the infantile female sheep.

Several intermediate forms in the processing of rat lutropin subunits as shown by sodium dodecyl sulphate polyacrylamide gel electrophoresis

To study the biosynthesis in situ of lutropin (LH) subunits, anterior pituitary cells in culture were employed. The cells were incubated in the presence of [35S]methionine. Labelled polypeptides, immunologically related to alpha and LH beta subunits, were isolated by specific immunoprecipitation, then analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and revealed by fluorography. Electrophoretic analysis of immunoprecipitates is a crucial step in this methodology. It permits simultaneous purification, characterization and accurate quantitation of the radioactivity specifically incorporated into LH subunits. Also, using SDS-PAGE, it is possible to isolate and identify the different processed forms of LH subunits.

Hormonal Regulation of Pituitary Gonadotropin Gene Expression

Lutropin (LH) and follitropin (FSH) are two members of a family of structurally related polypeptide hormones, that also includes pituitary thyrotropin (TSH) and placental human chorionic gonadotropin (hCG). Each hormone of this family contains two non identical, non covalently linked subunits, α and β. The primary structure of the a subunits is identical among hormones within a species, whereas the primary structures of the β subunits differ greatly and confer to the hormones their specific biological activities (Pierce and Parsons, 1981). Although they are not identical, the β subunits show enough homology in their primary structures to suggest that they arose by duplications and mutations of a single ancestral gene (Fontaine and Burzawa-Gerard, 1977).

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.