The G-protein coupled receptor GPR54 and its ligand, KiSS-1-derived peptide kisspeptin-54, appear to play an important role in the mechanism of puberty. This study measures the release of kisspeptin-54 in the stalk-median eminence (S-ME) during puberty and examines its potential role in the pubertal increase in LHRH-1 release in female rhesus monkeys. First, developmental changes in release of kisspeptin-54 and LHRH-1 were assessed in push-pull perfusate samples obtained from the S-ME of prepubertal, early pubertal, and midpubertal female rhesus monkeys. Whereas LHRH-1 levels in 10-min intervals had been measured previously for other experiments, kisspeptin-54 levels in 40-min pooled samples were newly measured by RIA. The results indicate that a significant increase in kisspeptin-54 release occurred in association with the pubertal increase in LHRH-1 release and that a nocturnal increase in kisspeptin-54 release was already observed in prepubertal monkeys and continued through the pubertal period. Second, we measured kisspeptin-54 release in the S-ME of midpubertal monkeys at 10-min intervals using a microdialysis method. Kisspeptin-54 release in the S-ME was clearly pulsatile with an interpulse interval of about 60 min, and approximately 75% of kisspeptin-54 pulses were correlated with LHRH-1 pulses. Finally, the effect of kisspeptin-10 on LHRH-1 release was examined with the microdialysis method. Kisspeptin-10 infusion through a microdialysis probe significantly stimulated LHRH-1 release in a dose-dependent manner. Collectively, the results are consistent with the hypothesis that kisspeptin plays a role in puberty.

Previously, we have reported that 17beta-estradiol (E(2)) induces an increase in firing activity of primate LH-releasing hormone (LHRH) neurons. The present study investigates whether E(2) alters LHRH release as well as the pattern of intracellular calcium ([Ca(2+)](i)) oscillations and whether G protein-coupled receptor 30 (GPR30) plays a role in mediating the rapid E(2) action in primate LHRH neurons. Results are summarized: 1) E(2), the nuclear membrane-impermeable estrogen, estrogen-dendrimer conjugate, and the plasma membrane-impermeable estrogen, E(2)-BSA conjugate, all stimulated LHRH release within 10 min of exposure; 2) whereas the estrogen receptor antagonist, ICI 182,780, did not block the E(2)-induced LHRH release, E(2) application to cells treated with pertussis toxin failed to induce LHRH release; 3) GPR30 mRNA was expressed in olfactory placode cultures, and GPR30 protein was expressed in a subset of LHRH neurons; 4) pertussis toxin treatment blocked the E(2)-induced increase in [Ca(2+)](i) oscillations; 5) knockdown of GPR30 in primate LHRH neurons by transfection with small interfering RNA (siRNA) for GPR30 completely abrogated the E(2)-induced changes in [Ca(2+)](i) oscillations, whereas transfection with control siRNA did not; 6) the estrogen-dendrimer conjugate-induced increase in [Ca(2+)](i) oscillations also did not occur in LHRH neurons transfected with GPR30 siRNA; and 7) G1, a GPR30 agonist, resulted in changes in [Ca(2+)](i) oscillations, similar to those observed with E(2). Collectively, E(2) induces a rapid excitatory effect on primate LHRH neurons, and this rapid action of E(2) appears to be mediated, in part, through GPR30.

To understand the mechanism of pulsatile luteinizing hormone-releasing hormone (LHRH) release, we examined whether cultured LHRH neurons exhibit spontaneous intracellular Ca(2+) ([Ca(2+)](i)) signaling. The olfactory placode and the ventral migratory pathway of LHRH neurons from rhesus monkey embryos at embryonic ages 35-37 were dissected out and cultured on glass coverslips. Two to five weeks later, cultured cells were labeled with fura-2 and examined for [Ca(2+)](i) signaling by recording changes in [Ca(2+)](i) every 10 sec for 30-175 min. Cells were fixed and immunostained for LHRH and neuron-specific enolase. In 20 cultures, 572 LHRH-positive cells exhibited [Ca(2+)](i) oscillations at an interpulse interval (IPI) of 8.2 +/- 0.7 min and a duration of 88.8 +/- 2.9 sec. LHRH-negative neurons in culture exhibited only occasional [Ca(2+)](i) oscillations. In 17 of 20 cultures with LHRH-positive cells, [Ca(2+)](i) oscillations occurred synchronously in 50-100% of the individual cells, whereas [Ca(2+)](i) oscillations in cells in the remaining three cultures did not synchronize. Strikingly, in 12 of 17 cultures the synchronization of [Ca(2+)](i) oscillations repeatedly occurred in complete unison at 52.8 +/- 3.0 min intervals, which is similar to the period observed for LHRH release, whereas in 5 of 17 cultures the less tight synchronization of [Ca(2+)](i) oscillations repeatedly occurred at 23.4 +/- 4.6 min intervals. IPI of [Ca(2+)](i) oscillations in cells with tight synchronization and less tight synchronization did not differ from IPI in cells without synchronization. The results indicate that LHRH neurons derived from the monkey olfactory placode possess an endogenous mechanism for synchronization of [Ca(2+)](i) oscillations. Whether synchronization of [Ca(2+)](i) oscillations relates to neurosecretion remains to be investigated.

Release of gonadotropin releasing hormone (GnRH) from the medial basal hypothalamus (MBH)/median eminence region (S-ME) is essential for normal reproductive function. GnRH release is profoundly regulated by the negative and positive feedback effects of ovarian estradiol (E 2 ). Here we report that neuroestradiol, released in the S-ME, also directly influences GnRH release in ovariectomized female monkeys, in which the ovarian source of E 2 is removed. We found that (1) brief infusion of E 2 benzoate (EB) to the S-ME rapidly stimulated release of GnRH and E 2 in the S-ME of ovariectomized monkeys, (2) electrical stimulation of the MBH resulted in GnRH release as well as E 2 release, and (3) direct infusion of an aromatase inhibitor to the S-ME suppressed spontaneous GnRH release as well as the EB-induced release of GnRH and E 2 . These findings reveal the importance of neuroestradiol as a neurotransmitter in regulation of GnRH release. How circulating ovarian E 2 interacts with hypothalamic neuroestrogens in the control of GnRH release remains to be investigated.

Feedback controls of estrogen in LHRH-1 neurons play a pivotal role in reproductive function. However, the mechanism of estrogen action in LHRH-1 neurons is still unclear. In the present study, the effect of estrogens on intracellular calcium ([Ca(2+)](i)) oscillations in primate LHRH-1 neurons was examined. Application of 17beta-estradiol (E(2), 1 nm) for 10 min increased the frequency of [Ca(2+)](i) oscillations within a few minutes. E(2) also increased the frequency of [Ca(2+)](i) synchronization among LHRH-1 neurons. Similar E(2) effects on the frequency of [Ca(2+)](i) oscillations were observed under the presence of tetrodotoxin, indicating that estrogen appears to cause direct action on LHRH-1 neurons. Moreover, application of a nuclear membrane-impermeable estrogen dendrimer conjugate, not control dendrimer, resulted in a robust increase in the frequencies of [Ca(2+)](i) oscillations and synchronizations, indicating that effects estrogens on [Ca(2+)](i) oscillations and their synchronizations do not require their entry into the cell nucleus. Exposure of cells to E(2) in the presence of the estrogen receptor antagonist ICI 182,780 did not change the E(2)-induced increase in the frequency of [Ca(2+)](i) oscillations or the E(2)-induced increase in the synchronization frequency. Collectively, estrogens induce rapid, direct stimulatory actions through receptors located in the cell membrane/cytoplasm of primate LHRH-1 neurons, and this action of estrogens is mediated by an ICI 182,780-insensitive mechanism yet to be identified.