GnRH-Stimulated Glycosylation (Proximal and Distal) of Luteinizing Hormone by Cultured Rat Pituitary Cells

Abstract

In the present work, the effects of GnRH on the translation (by [¹⁴C]leucine incorporation; [¹⁴C]Leu-LH) and the glycosylation of LH by rat pituitary cells in primary culture were established. The use of specific markers as radioactive precursors made it possible to discriminate the action of the neurohormone on proximal glycosylation (by [³H]mannose incorporation; [³H]Man-LH) as well as distal glycosylation (by [³H]galactose incorporation; [³H]Gal-LH) in the course of synthesis and release of LH. Pituitary cells from ovariectomized adult rats were incubated for different periods between 0 and 5 h in medium containing [¹⁴C]Leu plus [³H]Man or [¹⁴C]Leu plus [³H]Gal with or without 10 nM GnRH. GnRH increased synthesis and release of newly synthesized LH. The magnitude of the stimulatory effect on the kinetics of [¹⁴C]Leu (slope = 63.58; 158% of control) and [³H]Man (slope = 75.15; 161%) incorporation to LH was similar. The action of the neurohormone appears to be exerted on translation, the increased [³H]Man incorporation being a secondary phenomenon arising from the greater amount of available polypeptide chains as acceptors of the polymannose core. However, a direct effect of GnRH on proximal glycosylation cannot be excluded. GnRH also stimulated the kinetics of release of [¹⁴C]Leu-LH (slope = 6.14; 236% of control) and [³H]Man-LH (slope = 8.06; 191%). Comparatively, the effect of GnRH on [³H]Gal-LH was detected earlier than that on LH labeled with the other precursors; increases in rates of production (slope = 71.57; 278% of control) and release (slope = 32.08; 494%) were higher than those in [¹⁴C]Leu- and [³H]Man-LH kinetics, indicating that GnRH acts specifically on this distal step of LH glycosylation. GnRH enhanced the relative terminal glycosylation ([³H]Gal/[¹⁴C]Leu ratio) of total and released LH without modifying the relative proximal glycosylation ([³H]Man/[¹⁴C]Leu ratio) of the hormone. We conclude that GnRH can induce not only changes in the quantity (greater number of molecules) but also in the quality (molecules more glycosylated) of the secreted LH by acting directly at translation and distal glycosylation level.

Pituitary Luteinizing Hormone Microheterogeneity in the Afternoon of Proestrus in Rats: Some New Insights

“…In contrast, the present results show the relative increase in less alkaline LH species in the proestrus-afternoon pituitaries. These findings are consistent with those of our previous studies [11,12] showing that GnRH increased [ 3 H]galactose incorporation, by acting directly at the distal glycosylation level. In the case of pituitaries from proestrusafternoon rats, the shift toward isoforms with pI values in the range of 9.00-8.01 could potentially result from sialylation of increased terminal galactose.…”

“…In terms of endocrine regulation of LH isoforms, existing evidence suggests that both hypothalamic and gonadal inputs are involved not only in regulating synthesis and release of LH but also in modulating the molecular nature of the hormone [10,11].…”

“…In our previous studies [11,12] we presented evidence of the GnRH-stimulatory effect on terminal LH glycosylation in vitro as estimated by increased [ 3 H]Gal incorporation into newly synthesized hormone. On the basis of those findings, the aim of the present report was to determine possible modifications in pituitary LH isoforms induced by the in vivo increase in GnRH at the time of the preovulatory gonadotropin surge.…”

Thyrotropin-releasing hormone time-dependently influences thyrotropin microheterogeneity--an in vivo study in euthyroidism

“…Releasing hormones such as gonadotropin-releasing hormone are known to induce not only changes in the quantity (greater numbers of molecules) but also in the quality (differences in glycosylation) of the secreted glycoprotein hormone by acting directly at translation and distal glycosylation levels (Ulloa-Aguirre et al 1995, Perez & Apfelbaum 1996, Lambert et al 1998, Savastano et al 1998, Timossi et al 1998. As far as the pituitary-thyroid axis is concerned, the availability of an efficient immuno-affinity method for the purification and concentration of serum TSH molecules (Schaaf et al 1995, Persani et al 1998) allows separation of different TSH isohormones.…”

Naturally Occurring Follicle-Stimulating Hormone Glycosylation Variants

“…However, subsequent studies in rats indicated that estrogen inhibited pituitary expression of α 2-3-sialyltransferase [ 22 , 23 ], suggesting that α2-6-sialyltransferase activity increased to compensate for the loss of one isoform. GnRH was reported to increase galactose content of LH glycans [ 24 ], indicating increased branching. GnRH stimulation of human subjects resulted in release of less acidic forms of hFSH into the serum [ 25 , 26 ] and secretion of less acidic FSH forms from dispersed rat pituitary cells, even in the presence of estrogen [ 21 ].…”