The release and synthesis of LH and FSH were studied in adenohypophyses from ovariectomized rats incubated for a period of 4 h in flasks containing 1 ml Eagle's medium. One hemipituitary was used as the experimental gland and the other half served as a control. Glands from ovariectomized untreated animals showed a spontaneous release of LH and FSH and the amount of hormones released (per mg gland) by both the hemipituitaries was not significantly different. Also the content of the hormones at the end of the incubation period was similar in both halves. Gonadotrophin-releasing hormone (Gn-RH) added to the incubation medium stimulated the release of LH and FSH. A dose--response relationship was obtained between doses of 0-51 and 8-00 ng/ml medium. Although lower doses were required to increase the release of LH, the amount of FSH released was higher when expressed as a percentage of gland content. Pituitary glands from ovariectomized rats treated with 5 mug oestradiol benzoate 24 h before being killed showed an increase in sensitivity to Gn-RH, but the response decreased when oestrogen was injected 2 h before death. Also the addition of oestradiol-17beta to the incubation medium inhibited LH and FSH release induced by Gn-RH. Gonadotrophin-releasing hormone increased the spontaneous synthesis of LH and FSH observed in the incubated pituitaries. This effect of Gn-RH was stimulated by the injection of oestrogen into the donor animals whereas administration of oestrogen into the medium enhanced the synthesis of LH and partially inhibited that of FSH. These results provide evidence for a dual effect of oestrogen on the release of LH and FSH induced by Gn-RH. They also show that synthesis of gonadotrophic hormones was favoured by oestrogen or by increased gonadotrophin release.

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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.

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.

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.