Early prepubertal ontogeny of pulsatile gonadotropin-releasing hormone (GnRH) secretion: I. Inhibitory autofeedback control through prolyl endopeptidase degradation of GnRH.

GnRH[1-5], a subproduct resulting from degradation of GnRH by prolyl endopeptidase (PEP) and endopeptidase 24.15 (EP24.15) was known to account for an inhibitory autofeedback of GnRH secretion through an effect at the N-methyl-D-aspartate (NMDA) receptors. This study aimed at determining the possible role of such a mechanism in the early developmental changes in frequency of pulsatile GnRH secretion. Using retrochiasmatic explants from fetal male rats (day 20-21 of gestation), no GnRH pulses could be observed in vitro, whereas pulses occurred at a mean interval of 86 min from the day of birth onwards. This interval decreased steadily until day 25 (39 min), during the period preceding the onset of puberty. Based on GnRH[1-10] or GnRH[1-9] degradation and GnRH[1-5] generation after incubation with hypothalamic extracts, EP24.15 activity did not change with age, whereas PEP activity was maximal at days 5-10 and decreased subsequently until day 50. These changes were consistent with the ontogenetic variations in PEP messenger RNAs (mRNAs) quantitated using RT-PCR. Using fetal explants, the NMDA-evoked release of GnRH was potentiated in a dose-dependent manner by bacitracin, a competitive PEP inhibitor and the desensitization to the NMDA effect was prevented using 2 mM of bacitracin. At day 5, a higher bacitracin concentration of 20 mM was required for a similar effect. Pulsatile GnRH secretion from fetal explants was not caused to occur using bacitracin or Fmoc-Prolyl-Pyrrolidine-2-nitrile (Fmoc-Pro-PyrrCN), a noncompetitive PEP inhibitor. At postnatal days 5 and 15, a significant acceleration of pulsatility was obtained using 1 microM of Fmoc-Pro-PyrrCN or 2 mM of bacitracin. At 25 and 50 days, a lower bacitracin concentration of 20 microM was effective as well in increasing the frequency of GnRH pulsatility. We conclude that the GnRH inhibitory autofeedback resulting from degradation of the peptide is operational in the fetal hypothalamus but does not explain the absence of pulsatile GnRH secretion at that early age. After birth, PEP activity is high and may account for the low frequency of pulsatility. The potency of that effect decreases before the onset of puberty and may contribute to the acceleration of GnRH pulsatility.

Pulsatile secretion of gonadotropin-releasing hormone by rat hypothalamic explants without cell bodies of GnRH neurons [corrected].

Gonadotropin-releasing hormone (GnRH) is typically secreted in a pulsatile manner. It is still unclear whether pulsatility depends on a GnRH pulse generator residing in the GnRH neurons or in other neurons. Since the cell bodies of GnRH neurons are located rostrally to the optic chiasm and the majority of GnRH terminals in the median eminence of the rat hypothalamus, we have compared GnRH secretion from individual preoptic, retrochiasmatic and median eminence explants using a static incubation system. GnRH is released from the three different types of explant in response to depolarization with veratridine or glutamate receptor stimulation using the agonist N-methyl-D-aspartate. Only the retrochiasmatic explants, however, show a characteristic pulsatile secretion of GnRH. The mean (+/- SD) interpulse interval is respectively 37 +/- 5, 25 +/- 4 and 12 +/- 1 min when the fractions are collected at 7.5-, 5.0- and 2.5-min intervals. The immunocytochemically stained GnRH cell bodies are normally distributed in the preoptic explants (n = 212-420) while only 3 GnRH cell bodies can be visualized in 7 retrochiasmatic explants. Semi-quantitative RT-PCR shows that GnRH mRNA is present in the retrochiasmatic explant in a ratio of about 1:600 relative to the preoptic explant. We conclude that pulsatile GnRH secretion occurs in the virtual absence of GnRH cell bodies but does not occur from GnRH terminals in the isolated median eminence. These data further indicate that a mechanism of GnRH pulsatility is located in the retrochiasmatic hypothalamus and involves neurons different from the GnRH neurons.

Duality of glutamatergic and GABAergic control of pulsatile GnRH secretion by rat hypothalamic explants: I. Effects of antisense oligodeoxynucleotides using explants including or excluding the preoptic area.

Using antisense oligodeoxynucleotides we aimed to study the role of N-methyl-D-aspartate (NMDA) and gamma-aminobutyric acid (GABA) receptors in the mechanism of Gonadotrophin-releasing hormone (GnRH) secretion in vitro. Since GnRH cell bodies are located in the rat preoptic hypothalamus while most GnRH terminals are in the retrochiasmatic hypothalamus, we compared the effects of oligodeoxynucleotides on explants of the whole (preoptic area included) or retrochiasmatic hypothalamus. When GnRH secretion is evoked by muscimol and NMDA, a time-related reduction of GnRH secretion is caused by antisense oligodeoxynucleotides for the beta subunit of the GABAA receptor and the NR2A subunit of the NMDA receptor, respectively. After 6-7 h, binding studies of tritiated ligands show a decrease in GABA- and NMDA-receptor expression. While these antisense effects are observed using whole explants, no such effects are seen using retrochiasmatic explants, indicating that the facilitatory GABAA and NMDA receptors are encoded in the preoptic area. Using several missense oligodeoxynucleotides or antisense for the NR2B and NR2C subunits of the NMDA receptor, the muscimol- and NMDA-evoked release of GnRH is not affected. When spontaneous pulsatile GnRH secretion is studied, the NR2A antisense oligodeoxynucleotides cause an increase of the interpulse interval. This increase is seen using whole but not retrochiasmatic explants. In contrast, the GABAA and NR2C antisense oligodeoxynucleotides result in a reduction of GnRH interpulse interval. Such a reduction is seen using whole as well as retrochiasmatic explants, indicating that the GABAA and NMDA receptors which mediate inhibition of GnRH pulsatility are encoded in the retrochiasmatic hypothalamus. We conclude that NMDA receptors (NR2A subunit) encoded in the preoptic hypothalamus mediate a facilitatory effect on GnRH pulsatility while GABAA and NMDA (NR2C subunit) receptors encoded in the retrochiasmatic hypothalamus mediate an inhibition of GnRH pulsatility. Pulsatile GnRH secretion is affected differently than the agonist-evoked release of GnRH suggesting that the GnRH secretory neurons and the GnRH pulse generator consist of different cellular entities.

Duality of glutamatergic and GABAergic control of pulsatile GnRH secretion by rat hypothalamic explants: II. Reduced NR2C- and GABAA-receptor-mediated inhibition at initiation of sexual maturation.

N-methyl-D-aspartate (NMDA) receptors and gamma-aminobutyric acid (GABA) receptors are involved in the mechanism of pulsatile gonadotrophin-releasing hormone (GnRH) secretion. The aim of this study was to elucidate the role of those receptors in the acceleration of pulsatile GnRH secretion seen at onset of puberty. Using hypothalamic explants from prepubertal (15 days), early pubertal (25 days) and adult (50 days) male rats, we studied the effects of pharmacological antagonists and antisense oligodeoxynucleotides on GnRH release evoked by NMDA and GABA receptor agonists as well as the interval between spontaneous GnRH secretory pulses. At the three studied ages, the muscimol-evoked release of GnRh is similarly inhibited by the GABAA receptor antagonist bicuculline. In contrast, the frequency of pulsatility is stimulated by bicuculline as indicated by a reduction of the mean GnRh interpulse interval from 60 to 40 min and such an effect is seen at 15 days only. The GnRH interpulse interval is also reduced by GABAA receptor antisense oligodeoxynucleotides at 15 days while no effects are seen at 25 days. At the three studied ages, the NMDA-evoked release of GnRH and the GnRh interpulse interval are similarly inhibited by 100 or 500 microM of the NMDA receptor antagonist 7-chlorokynurenic acid (7CK). These effects are consistent with the increase of GnRH interpulse interval caused by NR2A antisense oligodeoxynucleotides at 15 days (86 vs 64 min in controls) as well as 25 days (44 vs 36 min). A low (5 microM) concentration of 7CK does not result in any effect except a reduction of GnRH interpulse interval which is seen at 15 days only. A similar reduction of GnRh interpulse interval is obtained using NR2C antisense oligodeoxynucleotides at 15 days (50 vs 64 min in controls) while no effects are seen at 25 days (35 vs 36 min). At 25 days, muscimol can prevent the developmental increase in frequency of pulsatile GnRH secretion. In summary, pulsatile GnRH secretion by the prepubertal hypothalamus characteristically involves an inhibition mediated through GABAA receptors and the NR2C subunit of NMDA receptors. Based on these data, we propose a model for the mechanism of the onset of puberty which involves the disappearance or inactivation of GABAergic neurons located in the retrochiasmatic hypothalamus and expressing the NR2C subtype of NMDA receptors.

Endogenous glutamate involvement in pulsatile secretion of gonadotropin-releasing hormone: evidence from effect of glutamine and developmental changes.

The secretion of GnRH can be stimulated by glutamate (GLU) and GLU agonists, whereas GLU receptor antagonists inhibit GnRH. Using 6-diazo-5-oxo-L-norleucine (DON), an inhibitor of glutaminase, we aimed to study the involvement of endogenous GLU in GnRH secretion through the effects of impaired GLU biosynthesis from its precursor glutamine (GLN). GnRH secretion by hypothalamic explants of male rats, aged 15 and 50 days, was compared, because the frequency of spontaneous GnRH secretory pulses showed a 2-fold increase between those two ages. Using explants of 50-day-old rats, GLN elicited GnRH secretion in a similar dose-related manner as GLU. DON prevented GLN-evoked secretion of GnRH, whereas the effect of GLU was not altered. DON also markedly inhibited spontaneous pulsatile secretion of GnRH and the secretory response to veratridine, a Na+ channel opener. The inhibitory effect of DON on veratridine-evoked secretion of GnRH was directly related to the duration of exposure to DON and the frequency of GnRH secretory episodes. Using explants of 15-day-old rats, GLN could elicit GnRH release, although this response was lower than GLU-evoked secretion of GnRH. The DON concentrations required for inhibition of veratridine-evoked secretion of GnRH were lower at 15 days than at 50 days. These data indicate that 1) GLU biosynthesis from GLN is a prerequisite to the physiological mechanism of pulsatile GnRH secretion; and 2) inhibition of veratridine- or GLN-induced secretion of GnRH requires higher DON concentrations after the onset of puberty than before. This suggests that glutaminase, the enzyme controlling GLU biosynthesis from GLN, shows increased activity after the onset of puberty when the frequency of pulsatile GnRH secretion is increased as well.