Neurokinin B stimulates GnRH release in the male monkey (Macaca mulatta) and is colocalized with kisspeptin in the arcuate nucleus

Abstract

Human genetics indicate that kisspeptin and neurokinin B (NKB) signaling are necessary for generating pulsatile LH release and therefore for initiation of puberty and maintaining gonadal function. In the present study, male monkeys were employed to examine 1) whether activation of the NKB receptor (NK3R) is associated with GnRH release, and 2) hypothalamic localization of these peptides using immunofluorescence histochemistry. Agonadal juveniles, in which pituitary responsiveness to GnRH was heightened by GnRH priming, were employed to indirectly examine GnRH-releasing actions of NK3R and kisspeptin receptor agonists by tracking LH after their i.v. injection. Castrated adults were used for immunohistochemistry. Single i.v. injections of NKB or senktide (an NK3R agonist) elicited robust LH discharges that were abolished by GnRH receptor antagonism (acyline) confirming the ligands' hypothalamic action. Intermittent infusion of senktide (1-min pulse every hour for 4 h), in contrast to that of kisspeptin, failed to sustain pulsatile GnRH release. Repetitive senktide injections did not compromise the GnRH-releasing action of kisspeptin. NKB and kisspeptin were colocalized in perikarya of the arcuate nucleus and in axonal projections to the median eminence, confirming earlier findings in sheep. These results are consistent with the human genetics, and indicate that although brief activation of NK3R stimulates GnRH release, repetitive stimulation of this pathway, in contrast to that of kisspeptin receptor, fails to sustain pulsatile GnRH release. In addition, the data provide a platform for future elucidation of the interactions between NKB and kisspeptin that are required for generating pulsatile GnRH release in primates.

Figure 1

Circulating concentrations of LH (mean ± sem) after bolus iv injections (arrows) of senktide (S) (50 μg), NKB (N) (100 μg), and GnRH (G) (0.3 μg) given at 2-h intervals to agonadal juvenile male monkeys. Note that the order of treatment with the three peptides was different for each of the three animals (see experiment 2 under experimental design in Materials and Methods), and for presentation, the responses of two animals have been reordered to match those for the animal that received the sequence S, N, and G. Also, note that LH values for the last four time points after GnRH represent data from two animals only. n = 3.

Figure 2

Circulating concentrations of LH (mean ± sem) after the last GnRH (G) pulse of the priming infusion (open arrow) and sequential bolus iv injections (arrows) of senktide (S) (50 μg), NKB (N) (100 μg), and GnRH (0.3 μg) (black arrows) given at 2-h intervals to agonadal juvenile male monkeys when a GnRH-R antagonist, acyline (A) (arrowhead), was given 30 min after the first GnRH challenge (closed data points). Note the absence of LH discharges in response to the three peptides after administration of acyline. Treatment with the vehicle for the GnRH-R antagonist (V) (5% mannitol in saline; arrowhead) did not interrupt the LH discharge induced by a bolus iv injection of senktide (open data points). The response to NKB and the second GnRH challenge was not studied after vehicle administration. n = 4.

Figure 3

Circulating concentrations of LH (mean ± sem) after bolus iv injections (arrows) of senktide (S) (50 μg), Kp-10 (K) (2 μg), and GnRH (G) (0.3 μg) given at 2-h intervals to agonadal juvenile male monkeys. Note that the order of treatment with the three peptides was different for each of the three animals (see experiment 4 under experimental design in Materials and Methods), and for presentation, the responses of two animals have been reordered to match those for the animal that received the sequence S, K, and G. n = 3.

Figure 4

Administration of a nonpeptide NK3R antagonist (SB222200; white arrow) at −15 min abolished the LH response to an iv bolus injection of senktide (S) (50 μg; black arrow, triangle data points) given at time 0 but did not interfere with LH release when an iv bolus of KP-10 (K) (2 μg; black arrow, closed circle data points) was given at time 0. Administration of vehicle (V) at −15 min did not interfere with senktide induced LH release (open data points). Mean LH concentrations (±sem) are shown. n = 3.

Figure 5

Discharges of LH, as reflected by circulating concentrations of the gonadotropin (mean ± sem), during the last priming pulse of GnRH (G) (broken black arrow) in agonadal male monkeys were sustained without decrement by an intermittent iv infusion of 2 μg Kp-10 administered as a 1-min pulse every hour for 5 h and initiated at time 0 (open data points) after concomitantly terminating the GnRH-priming infusion. In striking contrast, a similar intermittent iv infusion of senktide (50 μg/pulse) after termination of GnRH priming failed to mimic the action of repetitive Kp-10 administration. Instead, LH levels (closed data points) fell in parallel with those observed during intermittent iv infusion of vehicle (gray data points). The down-regulation of the LH response to repetitive senktide administration, however, did not compromise the GnRH-releasing action of an iv bolus of Kp-10 (K) (black arrow) administered after the last of four pulses of senktide. White block arrows indicate time of hourly intermittent infusions of Kp-10 (K) or senktide (S) or vehicle (V). Black arrow at 240 min indicates time of the last of 5 Kp-10 pulses or of the Kp-10 challenge after the intermittent infusion of either senktide or vehicle. n = 3.

Figure 6

Confocal immunofluorescence projections showing colocalization of NKB (red fluorescence, Cy3) and kisspeptin (green fluorescence, Alexa 488) positive perikarya and fiber networks in the arcuate nucleus (ARC) and median eminence (ME), respectively, of a castrated adult male rhesus monkey. A–C, Confocal projections (magnification, ×10; 1-μm optical sections) showing immunopositive kisspeptin (A) and NKB (B) profiles and their merged image (C) in a coronal hemisection taken through the ARC. The third ventricle (3V) is seen on the right hand side of the half section. Note that the colocalization of NKB with kisspeptin (yellow) in both ARC and ME. D and E, Higher magnification (×40, 1-μm optical sections) confocal projections taken from the ARC (D) and the ME (E) of the section shown in A–C. Note that the colocalization of NKB and kisspeptin in many of the ARC kisspeptin positive perikarya (D) and in kisspeptin axonal projections in the ME (E). Scale bar, 100 μm (A–C) and 20 μm (D and E).

Figure 7

Confocal dual immunofluorescence projections (magnification, ×10; 1-μm optical sections) of a pair of coronal hemi-MBH sections taken at the level of the arcuate nucleus (ARC)-median eminence (ME) region from each of four castrated adult male monkeys stained for NKB (red fluorescence, Cy3) and kisspeptin (green fluorescence, Alexa 488). The left hand section of each pair (A–D) is anterior with respect to the more caudal section shown on the right (A′–D′). Note that colocalization of the two peptides (yellow) in approximately 40–60% of kisspeptin positive neurons of the ARC. 3V, Third ventricle. Scale bar, 100 μm.

Figure 8

A and B, Confocal immunofluorescence projections showing the distribution of NKB fibers (red fluorescence, Cy3) in relation to GnRH axons (green fluorescence, Alexa 488) in coronal hemisections of the median eminence (ME) of two castrated adult male monkeys (magnification, ×10; 1-μm optical sections). A′ and B′, Confocal projections (magnification, ×100; 1-μm optical sections) of NKB-GnRH interactions in regions of the internal zone of the median eminence shown in the area outlined by the rectangles in A and B, respectively. Contacts between NKB and GnRH fibers (white arrows) were confirmed by examination of individual optical sections. 3V, Third ventricle; ARC, lateral arcuate nucleus. Scale bars, 100 μm (A and B) and 10 μm (A′ and B′).