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. 2013 Apr 2;110(14):5677-82.
doi: 10.1073/pnas.1213594110. Epub 2013 Mar 18.

Synchronous activation of gonadotropin-releasing hormone gene transcription and secretion by pulsatile kisspeptin stimulation

Han Kyoung Choe  1 , Hee-Dae Kim, Sung Ho Park, Han-Woong Lee, Jae-Yong Park, Jae Young Seong, Stafford L Lightman, Gi Hoon Son, Kyungjin Kim
Affiliations

Synchronous activation of gonadotropin-releasing hormone gene transcription and secretion by pulsatile kisspeptin stimulation

Han Kyoung Choe et al. Proc Natl Acad Sci U S A. .

Abstract

Pulsatile release of hypothalamic gonadotropin-releasing hormone (GnRH) is essential for pituitary gonadotrope function. Although the importance of pulsatile GnRH secretion has been recognized for several decades, the mechanisms underlying GnRH pulse generation in hypothalamic neural networks remain elusive. Here, we demonstrate the ultradian rhythm of GnRH gene transcription in single GnRH neurons using cultured hypothalamic slices prepared from transgenic mice expressing a GnRH promoter-driven destabilized luciferase reporter. Although GnRH promoter activity in each GnRH neuron exhibited an ultradian pattern of oscillations with a period of ∼10 h, GnRH neuronal cultures exhibited partially synchronized bursts of GnRH transcriptional activity at ∼2-h intervals. Surprisingly, pulsatile administration of kisspeptin, a potent GnRH secretagogue, evoked dramatic synchronous activation of GnRH gene transcription with robust stimulation of pulsatile GnRH secretion. We also addressed the issue of hierarchical interaction between the circadian and ultradian rhythms by using Bmal1-deficient mice with defective circadian clocks. The circadian molecular oscillator barely affected basal ultradian oscillation of GnRH transcription but was heavily involved in kisspeptin-evoked responses of GnRH neurons. In conclusion, we have clearly shown synchronous bursts of GnRH gene transcription in the hypothalamic GnRH neuronal population in association with episodic neurohormone secretion, thereby providing insight into GnRH pulse generation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Ultradian rhythm of GnRHp-dsLuc under basal conditions. (A) Real-time bioluminescence in GnRHp-dsLuc mice. Representative time-lapse images of a GnRH neuron are shown on the right. (B) Quantitative luminescence profile of a GnRH neuron shown in A. (C) Spontaneous synchronization of a GnRH neuronal population. Pulsatile peaks of 33 individual luciferase-positive cells in a POA culture are plotted. Each row represents an individual neuron (white dot, peak). Synchronization index (% Sync) is the percentage of luciferase-positive cells simultaneously reaching their peaks. Asterisks represent peaks identified by Cluster-8.
Fig. 2.
Fig. 2.
Synchronous activation of GnRH promoter activity and secretion by intermittent kisspeptin administration. (A) Time-lapse luminescence images of an individual GnRH neuron stimulated with intermittent kisspeptin pulses (10 nM, 15 min on, 45 min off for 6 h). Blue bar, kisspeptin administration. (B and C) Representative profiles of an individual GnRH neuron stimulated with vehicle (VEH, gray bar) or kisspeptin (KISS, blue bar) (continuous KISS, 2.5 nM; single or intermittent KISS, 10 nM; VEH, 0.1% distilled water). Data are shown as raw (B) or detrended profile (C). (D) Synchronization of GnRH neuronal population. Raster plot of normalized detrended luminescence of a representative batch is color-coded according to scale shown at left (H, high; L, low). Each row represents an individual GnRH neuron. % Sync represents the percentage of luciferase-positive cells simultaneously reaching their peaks. Gray line, typical range of synchronization under basal conditions. (E) GnRH secretion in perifused media (gray bar, VEH; blue bar, kisspeptin). For D–E, data are shown as the mean ± SEM (n = 61–92 cells from three to four batches per treatment). Asterisks represent peaks identified by Cluster-8.
Fig. 3.
Fig. 3.
GPR54–PKC pathway in kisspeptin-induced synchronous activation of GnRH transcription and secretion. (A) Representative profiles of an individual GnRH neuron stimulated with kisspeptin with vehicle or indicated antagonist (15a, 30 μM GPR54 antagonist; Gö 6983, 10 μM PKC inhibitor; VEH, 0.1% dimethyl sulfoxide). Asterisks represent peaks identified by Cluster-8. (B) Raster plot of GnRH promoter activity for a single POA culture. Each row represents an individual GnRH neuron. Normalized detrended values are color-coded according to the scale on left (H, high; L, low). % Sync is the percentage of luciferase-positive cells simultaneously reaching their peaks. (C) GnRH secretion in perifused media (blue bar, kisspeptin pulse; line, vehicle or indicated drug). (D) Peak value of synchronization. Two-way repeated measures analysis of variance (RM ANOVA), P < 0.01 (pharmacological agents), P < 0.01 (number of kisspeptin pulses), P < 0.01 (interaction). (E) Peak value of GnRH secretion. Two-way RM ANOVA, P < 0.01 (pharmacological agents), P < 0.01 (number of kisspeptin pulses), P = 0.9710 (interaction). In D and E, *P < 0.05; **P < 0.01 vs. VEH, Bonferroni posttest. (F) Cumulative GnRH secretion during 6 h after initiation of kisspeptin, **P < 0.01 vs. VEH, t test. In B–F, data are shown as mean ± SEM (n = 52–72 cells from three to four batches per treatment).
Fig. 4.
Fig. 4.
Effect of de novo protein synthesis and secretory pathway. (A) Representative profiles of an individual GnRH neuron with pulsatile kisspeptin stimulation with vehicle or indicated drug (BFA, brefeldin A, 10 μg/mL; CET, cetrorelix, 10 μM; CHX, cycloheximide, 100 μM; VEH, 0.1% dimethyl sulfoxide). Asterisks represent peaks identified by Cluster-8. (B) Synchronization of GnRH neuronal population. Raster plot of normalized detrended luminescence of a single POA culture is shown as a pseudocolor scale (H, high; L, low). Each row represents an individual GnRH neuron. % Sync is the percentage of luciferase-positive cells simultaneously reaching their peaks. (C) GnRH secretion in perifused media (blue bar, kisspeptin; line, vehicle or indicated drugs). (D) Peak value of synchronization. Two-way RM ANOVA, P < 0.01 (inhibitors), P < 0.01 (number of kisspeptin pulses), P < 0.01 (interaction). (E) Peak value of GnRH secretion. Two-way RM ANOVA, P < 0.05 (inhibitors), P < 0.01 (number of kisspeptin pulses), P = 0.1977 (interaction). In D and E, *P < 0.05, **P < 0.01 vs. VEH, Bonferroni posttest. (F) Cumulative GnRH secretion during 6 h after initiation of kisspeptin. **P < 0.01 vs. VEH, t test. In B–F, data are shown as mean ± SEM (n = 65–89 cells from three to five batches per treatment).
Fig. 5.
Fig. 5.
Ultradian rhythm of GnRHp-dsLuc and kisspeptin-induced synchronization in Bmal1 knock-out mice. (A) Representative luminescence profiles under basal conditions in POA cultures obtained from GnRHp-dsLuc in WT or Bmal1−/− (Bmal1 KO) background. Asterisks represent peaks identified by Cluster-8. (B) Synchronization of GnRH neuronal population derived from WT or Bmal1 KO mouse stimulated with kisspeptin. Raster plot of normalized detrended luminescence for a representative POA culture is shown as a pseudocolor scale (H, high; L, low). Each row represents an individual GnRH neuron. % Sync is the percentage of luciferase-positive cells simultaneously reaching their peaks. (C) GnRH secretion in perifused media (blue bar, kisspeptin pulse). (D) Peak value of synchronization. Two-way RM ANOVA, P < 0.01 (genotype), P < 0.01 (number of kisspeptin pulses), P = 0.7842 (interaction). (E) Peak value of GnRH secretion. Two-way RM ANOVA, P < 0.01 (genotype), P = 0.1245 (number of kisspeptin pulses), P = 0.6422 (interaction). In D and E, *P < 0.05 vs. VEH, Bonferroni posttest. (F) Cumulative GnRH secretion during 6 h after initiation of kisspeptin. *P < 0.05 vs. VEH, t test. In B–F, data are presented as mean ± SEM (n = 81–93 cells from four batches per genotype).
Fig. 6.
Fig. 6.
A hypothetical model for GnRH pulse generator synchronized by intermittent kisspeptin stimulation. (A) GnRH neuron, which secretes GnRH neurohormone into the hypothalamic–pituitary portal vessels, is innervated by kisspeptinergic neurons; cell bodies are located in the anteroventral periventricular nucleus (AVPV) or arcuate nucleus (ARC) of the hypothalamus. (B) Under basal conditions, GnRH gene expression is sporadic (Left). Pulsatile stimulation of kisspeptin dramatically recruits a subset of GnRH neurons to synchronize GnRH transcription with episodic GnRH secretion (Right). Each circle represents a single GnRH neuron residing in the hypothalamic neural network. Closed circles, GnRH neurons in sync; open circles, GnRH neurons out of sync. De novo protein synthesis and secretory pathway may mediate coupled bursting of GnRH transcription and secretion.
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