Marta E. Apfelbaum

Basal and GnRH-Induced Release and Synthesis of LH and FSH from Incubated Pituitary Glands throughout the 4-Day Estrous Cycle of the Rat

A comparative study of the patterns of LH and FSH released and synthesized during the normal estrous cycle of the rat was performed in vitro. Groups of female rats were killed at 09.00 h and 15.00 h throughout the 4-day estrous cycle and the adenohypophysis incubated for 4 h. Pituitary extracts and media were assayed for LH and FSH by radioimmunoassay. Both hormones exhibited maximum concentrations in tissue and medium on proestrus afternoon. The increased release of LH occurred during the day of proestrus, while that of FSH lasted from the afternoon of proestrus to the morning of estrus. Synthesis of LH exhibited a marked rising phase from the afternoon of diestrus-2 through the morning of estrus. When release and synthesis of LH and FSH were expressed as percentages of hormone concentration at the beginning of the incubation period, the percentages of FSH were greater than those of LH at all stages of the cycle. Addition of synthetic gonadotrophin-releasing hormone (GnRH) to the incubation medium was followed by a dose-dependent increase in release and synthesis of both LH and FSH. Pituitary responsiveness to GnRH reached a peak on the afternoon of proestrus and fell to a minimum during diestrus. Whereas maximum LH responsiveness correlated with the proestrus discharge of the hormone , that of FSH ceased before the basal release of the hormone, which terminates on estrus. The possible role of sex steroid hormones in regulating in vitro secretion of gonadotrophins and pituitary sensitivity to exogenous GnRH is discussed.

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

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 release LH without modifying the relative proximal glycosylation ([3H]Man/[14C]Leu ration) 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.

Role of Vasoactive Intestinal Peptide and 5-HT2 Receptor Subtype in Serotonin Stimulation of Basal and Thyrotropin-Releasing-Hormone- Induced Prolactin Release in vitro from Rat Pituitary Cells

We have previously demonstrated that 5-HT stimulates not only basal but also thyrotropin-releasing-hormone (TRH)-induced prolactin (PRL) release by acting directly at the pituitary gland level. In the present report, the participation of an autoparacrine action of VIP in the stimulatory effects of 5-HT and the involvement of the 5-HT2 receptor type in mediating serotonin-induced PRL release have been examined. Cultured anterior pituitary cells from ovariectomized adult rats were incubated for 1 h in 1 ml of T3-supplemented medium with or without the test substances. The results obtained in the presence of T3 confirm our previous observations, since treatment of the cells with 5-HT caused dose-dependent increases in basal PRL release, with an approximate EC50 of 3.68 × 10–8 M, and led to a significant potentiation (1.3-fold) of the TRH-induced PRL release. In order to evaluate the possible participation of vasoactive intestinal peptide (VIP) as mediator of the effects of 5-HT on PRL release, cells were incubated in the presence of 5-HT alone (3–1,000 nM) or 100 nM 5-HT plus 30 nM TRH, with or without 200 nM VIP antagonist (VIP-At): [D,4-Cl-Ph6,Leu17]VIP. VIP-At partially inhibited the release of PRL induced by 5-HT, both basal and TRHstimulated release. The stimulatory effect of 5-HT, however, was not eliminated by VIP-At, since the PRL released in response to 5-HT was still over the respective control ones. These results further support the findings suggesting that 5-HT acts directly at pituitary level by stimulating PRL release. Addition of the 5-HT2 receptor antagonist, ketanserin (1 µM) into the incubation medium resulted in the loss of cellular responsiveness to 5-HT, preventing not only the stimulatory effect of 5-HT on the basal but also on the TRH-induced PRL release. In conclusion, the results further strengthen the possibility that 5-HT increases the basal PRL release and potentiates the stimulatory effect of TRH by acting directly at the level of the lactotropes. These effects are not simply a consequence of autoparacrine action of VIP. In addition, it was shown that ketanserin completely antagonizes PRL response to 5-HT, indicating the involvement of the 5-HT2 receptor type in mediating PRL release.

Basal and gonadotrophin-releasing hormone-induced biosynthesis and release of luteinizing hormone: Effect of calcium deprivation

The present study examines the basal and gonadotrophin-releasing hormone (GnRH)-stimulated biosynthesis and release of luteinizing hormone (LH) by pituitary cells in primary culture, and the effect of extracellular calcium deprivation on these events. Pituitaries from ovariectomized adult rats were enzymatically dispersed and cultured for 96 h. The cells were then incubated for 5 h (Expts. 1 and 3) or for different time intervals between 0 and 5 h (Expt. 2), in medium containing [14C]leucine ([14C]leu) and [3H]glucosamine ([3H]gln), with or without GnRH. Total immunoreactive LH (iLH) was measured in the medium and the cell extract by radioimmunoassay. LH translation (as estimated by [14C]leu incorporation into LH; [14C]LH) and LH glycosylation (as estimated by [3H]gln incorporation into LH; [3H]LH) were measured by immunoprecipitation with specific LH beta antiserum in both medium and cell extract. Treating the cells with GnRH caused both time- and dose-dependent increases of iLH in the medium as well as in total (cells plus medium) content, with an approximate ED50 of 0.7 nM. GnRH also stimulated LH biosynthesis by increasing both LH polypeptide chain synthesis and LH glycosylation. The effect of GnRH on LH glycosylation was detected earlier than that on translation, the [3H]LH rates of production and release being higher than those of [14C]LH. These findings suggest that GnRH-induced translation and glycosylation of LH are independently regulated. Removal of extracellular calcium resulted in the loss of cellular responsiveness to GnRH, preventing not only the stimulatory effects of GnRH on total and released iLH but also the GnRH-induced incorporation of both [14C]leu and [3H]gln into newly synthesized LH. These observations suggest that GnRH-stimulated LH glycosylation and LH translation involve calcium-dependent mechanisms. Neither the uptake of radiolabeled precursors nor their incorporation into total protein were affected by GnRH or Ca(2+)-deficient (no added calcium) medium. The results also suggest that the release of newly synthesized LH is regulated differently from previously synthesized stored hormone.

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

Objective: The aim of the present report was to determine the possible modifications in rat pituitary LH isoforms induced by the spontaneous increase in GnRH at the time of the preovulatory gonadotropin surge. Design: The changes in the quantitative pattern and relative proportions of pituitary LH isoforms in rats on the afternoon of proestrus [INT-P(PM)] were evaluated by comparison with other stages of the estrous cycle (diestrus-1, diestrus-2 and estrus) and ovariectomized (7 and 30 days earlier) animals killed in the morning and in the afternoon of the corresponding day. Methods: The chromatofocusing technique (pH gradient 11.00–7.00) was used to analyze the different molecular species of intrapituitary LH. Results: Pituitary LH from diestrus-1 animals, considered as a baseline pattern in the cycling rat, eluted as 11 isoforms distributed in pH 9.62–8.82, with greater percentages in pH 9.50–9.01. Except for INT-P(PM) pituitaries, there were no major differences in the pattern of LH heterogeneity in the pituitaries of rats from various stages of the cycle. In contrast, significant changes in the charge distribution and relative abundance of LH isoforms were found in the pituitaries from INT-P(PM) rats. INT-P(PM) pituitaries resolved in 16 LH isoforms with a significant shift to less alkaline pIs (pH 9.62–8.11), the more abundant being focused within pH 9.00–8.51. Conversely, a shift to more basic isoforms resulted after ovariectomy, leading to the accumulation of less mature isoforms in the gonadotrope. Conclusions: Presumably, the use of animals on INT-P(PM), at the time of the preovulatory LH surge, made it possible to discriminate such changes in LH isoform distribution. That GnRH, released in association with the rising phase of the LH surge, induces these changes in pituitary LH polymorphism appears to be the most likely possibility. In a previous study we demonstrated that GnRH stimulated galactose incorporation into LH in vitro. In the case of pituitaries from INT-P(PM) rats, the shift toward less alkaline isoforms could potentially result from sialylation of increased terminal galactose.

Effect of cycloheximide and tunicamycin on the gonadotrophin-releasing hormone stimulated distal glycosylation of luteinizing hormone by rat pituitary cells

We have previously demonstrated that gonadotrophin-releasing hormone (GnRH) induces not only changes in quantity but also in quality on secreted luteinizing hormone (LH), by increasing [14C]Leu (translation) and [3H]Gal (distal glycosylation) incorporation into newly synthesized hormone. In the present report, we have further examined the GnRH-induced [3H]Gal-LH synthesis and release by treating anterior pituitary cells with polypeptide synthesis and glycosylation inhibitors (cycloheximide and tunicamycin, respectively). Pituitary cells from ovariectomized adult rats were cultured for 4 days and then incubated for different periods (0-5 h) in medium containing [14C]Leu plus [3H]Man or [14C]Leu plus [3H]Gal in the absence (basal) or presence of 10 nmol/L GnRH with or without (control) cycloheximide (1.0 and 4.0 microg/mL) or tunicamycin (0.5 and 2.0 microg/mL). At the end of each incubation period, the cells and the medium were separated and processed for DNA uptake and newly synthesized LH (labeled LH, by immunoprecipitation with a-betaLH) determinations. The velocity of synthesis and release (between 0 and 2 h, and between 2 and 5 h) was calculated by regression analysis and the statistical significance of differences was determined by the slope test. GnRH enhanced the rates of synthesis and release of [14C]Leu-, [3H]Man-, and [3H]Gal-LH to 157 and 237; 164 and 190; and 272 and 508% of basal values, respectively. Cycloheximide totally blocked synthesis and release of [14C]Leu-LH and greatly reduced those of [3H]Man-LH, resulting in the loss of cellular responsiveness to GnRH. Addition of tunicamycin to the pituitary cells inhibited the rates of synthesis and release of [3H]Man-LH which had been induced by GnRH, without altering those of [14C]Leu-LH. These findings indicate that glycosylation is not a condition for GnRH-stimulated LH translation. The GnRH-increased [3H]Gal-LH rates of synthesis and release were affected to a lesser extent by the inhibitors. Thus, GnRH stimulation of distal glycosylation can occur, albeit at a reduced rate, even though protein synthesis and glycosylation are blocked. In conclusion, the present results corroborate that GnRH stimulates the addition of galactose residues into LH molecule. This effect is not simply the consequence of stimulating LH polypeptide chain synthesis. In addition, it is shown that GnRH-increased LH translation is independent of glycosylation.

Rates of proximal and distal glycosylation of luteinizing hormone by cultured rat pituitary cells.

The purpose of the present work was to study the rate of basal luteinizing hormone (LH) glycosylation, discriminating the co-translational (proximal) and the post-translational (distal) glycosylation. The experiments were performed to determine the temporal relationship between the biosynthesis of the peptide chains (by [14C]leucine incorporation: [14C]Leu-LH) and the proximal (by [3H]mannose incorporation: [3H]Man-LH) and distal (by [3H]galactose incorporation: [3H]Gal-LH) glycosylation of LH, by rat pituitary cells in primary culture. In addition, the effects of cycloheximide (translation inhibitor) and tunicamycin (glycosylation inhibitor) on the rates of synthesis and release of [3H]Man-LH and [3H]Gal-LH were studied. The rates of synthesis and release (between 0 and 2 h and between 2 and 5 h) were calculated by regression analysis and the statistical significance of differences was determined by the slope test. The rate of synthesis of [3H]Man-LH (slope = 45.59) parallelled that of [14C]Leu-LH (slope = 41.39), which was in agreement with the assumption that the addition of the high mannose core is a co-translational event. Release of [3H]Man-LH (slope = 4.32) as well as that of [14C]Leu-LH (slope = 2.53) showed a lag period of approximately 2 h. The dynamics of [3H]Gal-LH secretion over the course of incubation, with a slower rate of synthesis (slope = 26.40) and a faster rate of release (slope = 6.34), differed from that of [3H]Man-LH. LH labeled with [3H]Gal was released from the early times of the incubation, indicating that galactose is added in the final stages of the secretory process into LH molecules, which are immediately released. LH translation blockage induced by cycloheximide was associated with a corresponding decrease of [3H]Man incorporation. On the other hand, addition of tunicamycin to the pituitary cells inhibited the rates of synthesis and release of [3H]Man-LH, without affecting those of [14C]Leu-LH. These findings show that proximal glycosylation depends on synthesis of the peptide chains, whereas the addition of the polymannose core is not a condition for translation. The rates of synthesis and release of [3H]Gal-LH were less affected by the antibiotics, and the inhibition was only significant at higher doses and long-time treatments. The present results demonstrate the independence of both steps of LH glycosylation, the rate of [3H]Gal-LH synthesis being 1.7-fold slower and that of release 1.5-fold faster than those of [3H]Man-LH, respectively. The data also suggest that glycosylation is not an essential step in the LH secretory process since the hormone, which is normally secreted in a glycosylated form, was synthesized, transported, and released without the carbohydrate side chains.