Kim L. Keen

An Increase in Kisspeptin-54 Release Occurs with the Pubertal Increase in Luteinizing Hormone-Releasing Hormone-1 Release in the Stalk-Median Eminence of Female Rhesus Monkeys in Vivo

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.

Involvement of G Protein-Coupled Receptor 30 (GPR30) in Rapid Action of Estrogen in Primate LHRH Neurons

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.

Effects of pulsatile infusion of the GABA(A) receptor blocker bicuculline on the onset of puberty in female rhesus monkeys.

In order to test the hypothesis that GABA is an inhibitory neurotransmitter restricting the release of LHRH before puberty, we examined the effects of pulsatile infusion of the GABA(A) receptor blocker, bicuculline, on the timing of puberty. Eleven female monkeys at 14-15 months of age were implanted with a stainless steel cannula into the base of the third ventricle above the median eminence. Five monkeys received bicuculline infusion every 2 h at a dose of 1 microM with a gradual increase to 100 microM in 10 microl using a portable infusion pump. The remaining 6 monkeys received similar infusions of saline. An additional 11 colony monkeys without cannula implantation were used for controls. Results indicate that bicuculline infusion advances the timing of puberty. The age of menarche (17.8+/-0.5 months) in the bicuculline infusion animals was significantly earlier than that in the saline controls (28.2+/-2.3, P < 0.001) as well as in colony controls (30.6+/-0.9, P < 0.001). The age of first ovulation (30.5+/-3.3 months) in bicuculline-treated animals was much younger (P < 0.001) than that in both controls (44.8+/-1.8 and 44.7+/-1.2, respectively). Bicuculline also accelerated the growth curve. These results suggest that the reduction of tonic GABA inhibition of LHRH neurons advances the onset of puberty.

Developmental Changes in GnRH Release in Response to Kisspeptin Agonist and Antagonist in Female Rhesus Monkeys (Macaca mulatta): Implication for the Mechanism of Puberty

Kisspeptin (KP) and KP-1 receptor (KISS1R) have emerged as important upstream regulators in the control of puberty. However, how developmental changes in KP-KISS1R contribute to the pubertal increase in GnRH release still remains elusive. In this study, we examined the effects of the KP agonist, human KP-10 (hKP-10), and the KP antagonist, peptide 234, on in vivo GnRH release in prepubertal and pubertal ovarian-intact female rhesus monkeys using a microdialysis method. We found that direct infusion of hKP-10 into the medial basal hypothalamus and stalk-median eminence region stimulated GnRH release in a dose-responsive manner, whereas infusion of peptide 234 suppressed GnRH release in both developmental stages. Because ovarian steroid feedback on GnRH release becomes prominent after the initiation of puberty in primates, we further examined whether ovarian steroids modify the GnRH response to hKP-10. Results demonstrate that the hKP-10-induced stimulation of GnRH release was eliminated by ovariectomy in pubertal, but not prepubertal, monkeys. Furthermore, replacement of estradiol into ovariectomized pubertal monkeys resulted in a partial recovery of the hKP-10-induced GnRH release. Collectively, these results suggest that a KISS1R-mediated mechanism, in addition to the pubertal increase in KP-54 release we previously reported, contributes to the pubertal increase in GnRH release and that there is a switch from an ovarian steroid-independent to -dependent mechanism in the response of GnRH to KP.

Rapid action of oestrogen in luteinising hormone-releasing hormone neurones: the role of GPR30.

Previously, we have shown that 17β‐oestradiol (E2) induces an increase in firing activity and modifies the pattern of intracellular calcium ([Ca2+]i) oscillations with a latency < 1 min in primate luteinising hormone‐releasing hormone (LHRH) neurones. A recent study also indicates that E2, the nuclear membrane impermeable oestrogen, oestrogen‐dendrimer conjugate, and the plasma membrane impermeable oestrogen, E2‐BSA conjugate, all similarly stimulated LHRH release within 10 min of exposure in primate LHRH neurones, indicating that the rapid action of E2 is caused by membrane signalling. The results from a series of studies further suggest that the rapid action of E2 in primate LHRH neurones appears to be mediated by GPR30. Although the oestrogen receptor antagonist, ICI 182, 780, neither blocked the E2‐induced LHRH release nor the E2‐induced changes in [Ca2+]i oscillations, E2 application to cells treated with pertussis toxin failed to result in these changes in primate LHRH neurones. Moreover, knockdown of GPR30 in primate LHRH neurones by transfection with human small interference RNA for GPR30 completely abrogated the E2‐induced changes in [Ca2+]i oscillations, whereas transfection with control siRNA did not. Finally, the GPR30 agonist, G1, resulted in changes in [Ca2+]i oscillations similar to those observed with E2. In this review, we discuss the possible role of G‐protein coupled receptors in the rapid action of oestrogen in neuronal cells.

Recent Discoveries on the Control of Gonadotrophin-Releasing Hormone Neurones in Nonhuman Primates

Since Ernst Knobil proposed the concept of the gonadotrophin‐releasing hormone (GnRH) pulse‐generator in the monkey hypothalamus three decades ago, we have made significant progress in this research area with cellular and molecular approaches. First, an increase in pulsatile GnRH release triggers the onset of puberty. However, the question of what triggers the pubertal increase in GnRH is still unclear. GnRH neurones are already mature before puberty but GnRH release is suppressed by a tonic GABA inhibition. Our recent work indicates that blocking endogenous GABA inhibition with the GABAA receptor blocker, bicuculline, dramatically increases kisspeptin release, which plays an important role in the pubertal increase in GnRH release. Thus, an interplay between the GABA, kisspeptin, and GnRH neuronal systems appears to trigger puberty. Second, cultured GnRH neurones derived from the olfactory placode of monkey embryos exhibit synchronised intracellular calcium, [Ca2+]i, oscillations and release GnRH in pulses at approximately 60‐min intervals after 14 days in vitro (div). During the first 14 div, GnRH neurones undergo maturational changes from no [Ca2+]i oscillations and little GnRH release to the fully functional state. Recent work also shows GnRH mRNA expression increases during in vitro maturation. This mRNA increase coincides with significant demethylation of a CpG island in the GnRH 5′‐promoter region. This suggests that epigenetic differentiation occurs during GnRH neuronal maturation. Third, oestradiol causes rapid, direct, excitatory action in GnRH neurones and this action of oestradiol appears to be mediated through a membrane receptor, such as G‐protein coupled receptor 30.

Tonic Control of Kisspeptin Release in Prepubertal Monkeys: Implications to the Mechanism of Puberty Onset

Previously we have shown that a reduction in γ-amino butyric acid (GABA) inhibition is critical for the mechanism initiating puberty onset because chronic infusion of the GABA(A) receptor antagonist, bicuculline, significantly increased GnRH release and accelerated the timing of menarche and first ovulation in female rhesus monkeys. Because previous studies in our laboratory indicate that in prepubertal female monkeys, kisspeptin release in the medial basal hypothalamus is low, whereas kisspeptin-10 can stimulate GnRH release, we hypothesized that a low level of kisspeptin release prior to puberty onset is due to tonic GABA inhibition. To test this hypothesis we examined the effects of bicuculline infusion on kisspeptin release using a microdialysis method. We found that bicuculline at 1 μM dramatically stimulates kisspeptin release in the medial basal hypothalamus of prepubertal monkeys but had little effect on kisspeptin release in midpubertal monkeys. We further examined whether bicuculline-induced GnRH release is blocked by the presence of the kisspeptin antagonist, peptide 234. We found that inhibition of kisspeptin signaling blocked the bicuculline-induced stimulation of GnRH release, suggesting that kisspeptin neurons may relay inhibitory GABA signals to GnRH neurons. This implies that a reduction in tonic GABA inhibition of GnRH release is, at least in part, mediated through kisspeptin neurons.

Developmental Increase in Kisspeptin-54 Release in Vivo Is Independent of the Pubertal Increase in Estradiol in Female Rhesus Monkeys (Macaca mulatta)

Kisspeptin (KP) signaling has been proposed as an important regulator in the mechanism of puberty. In this study, to determine the role of KP in puberty, we assessed the in vivo release pattern of KP-54 from the basal hypothalamus/stalk-median eminence in prepubertal and pubertal ovarian-intact female rhesus monkeys. We found that there was a developmental increase in mean KP-54 release, pulse frequency, and pulse amplitude, which is parallel to the developmental changes in GnRH release that we previously reported. Moreover, a nocturnal increase in KP-54 release becomes prominent after the onset of puberty. Because the pubertal increase in GnRH release occurs independent of the pubertal increase in circulating gonadal steroids, we further examined whether ovariectomy (OVX) modifies the release pattern of KP-54. Results show that OVX in pubertal monkeys enhanced mean KP-54 release and pulse amplitude but not pulse frequency, whereas OVX did not alter the release pattern of KP-54 in prepubertal monkeys. Estradiol replacement in OVX pubertal monkeys suppressed mean KP-54 release and pulse amplitude but not pulse frequency. Estradiol replacement in OVX prepubertal monkeys did not alter the KP-54 release pattern. Collectively these results suggest that the pubertal increase in KP release occurs independent of the pubertal increase in circulating estradiol. Nevertheless, the pubertal increase in KP release is not likely responsible for the initiation of the pubertal increase in GnRH release. Rather, after puberty onset, the increase in KP release contributes to further increase GnRH release during the progression of puberty.

Body Weight Impact on Puberty: Effects of High-Calorie Diet on Puberty Onset in Female Rhesus Monkeys

Secular trends toward a declining age at puberty onset with correlated changes in body weight have been reported in economically advanced countries. This has been attributed to excess calorie intake along with reduced physical activity in children. However, because the timing of puberty in humans is also influenced by other factors, such as genetic traits, living conditions, geographical location, and environmental chemicals, it is difficult to distinguish the effect of diet and body size from other factors in a human population. Here we report that feeding juvenile female rhesus monkeys born and raised at the Wisconsin National Primate Research Center with a high-calorie diet results in acceleration of body growth and precocious menarche. The monkeys fed a high-calorie diet also had an elevated body mass index. The most significant treatment effects on circulating hormones were increased leptin and IGF-I levels throughout the experiment. The findings of this study suggest the importance of close monitoring of juvenile feeding behaviors as an important intervention to reduce the prevalence of precocious development and metabolic diseases in adulthood.

STX, a novel nonsteroidal estrogenic compound, induces rapid action in primate GnRH neuronal calcium dynamics and peptide release

Previously, we reported that 1 nM 17ß-estradiol (E(2)) induces a rapid action, which is, in part, mediated through the G protein-coupled receptor GPR30 in primate GnRH neurons. Because it has been reported that the diphenylacrylamide compound, STX, causes estrogenic action in the mouse and guinea pig hypothalamus, the present study examined effects of STX in primate GnRH neurons and whether there is an action independent of GPR30. Results are summarized as follows. STX (10 nM) exposure increased 1) the oscillation frequency of intracellular calcium concentration ([Ca(2+)](i)), 2) the percentage of cells stimulated, and 3) the synchronization frequency of [Ca(2+)](i) oscillations. STX (10-100 nM) also stimulated GnRH release. The effects of STX on both [Ca(2+)](i) oscillations and GnRH release were similar to those caused by E(2) (1 nM), although with less magnitude. STX (10 nM)-induced changes in [Ca(2+)](i) oscillations were not altered by GPR30 small interfering RNA transfection, indicating that STX-sensitive receptors differ from GPR30. Finally, a higher dose of E(2) (10 nM) induced a larger change in [Ca(2+)](i) oscillations than that with a smaller dose of E(2) (1 nM), and the effects of 10 nM E(2) were reduced but not completely blocked by GPR30 small interfering RNA transfection, indicating that the effects of 10 nM E(2) in primate GnRH neurons are mediated by multiple membrane receptors, including GPR30 and STX-sensitive receptors. Collectively, the rapid action of E(2) mediated through GPR30 differs from that mediated through STX-sensitive receptors. The molecular structure of the STX-sensitive receptor remains to be identified.