Evidence from the agonadal juvenile male rhesus monkey (Macaca mulatta) for the view that the action of neurokinin B to trigger gonadotropin-releasing hormone release is upstream from the kisspeptin receptor

Abstract

Human genetics have revealed that kisspeptin signaling and neurokinin B (NKB) signaling are both required for robust pulsatile gonadotropin-releasing hormone (GnRH) release, and therefore for puberty and maintenance of adult gonadal function. How these two peptides interact to affect GnRH pulse generation remains a mystery. To address the hierarchy of the NKB and kisspeptin signaling pathways that are essential for GnRH release, two experiments were conducted using agonadal, juvenile male monkeys. Pituitary responsiveness to GnRH was first heightened by a pulsatile GnRH infusion to use the in situ pituitary as a bioassay for GnRH release. In the first experiment (n = 3), the kisspeptin receptor (KISS1R) was desensitized by a continuous 99-hour i.v. infusion of kisspeptin-10 (100 μg/h). During the last 4 h of continuous kisspeptin-10 infusion, desensitization of KISS1R was confirmed by failure of an i.v. bolus of kisspeptin-10 to elicit GnRH release. Desensitization of KISS1R was associated with a markedly blunted GnRH response to senktide. The response to senktide was progressively restored during the 72 h following termination of continuous kisspeptin-10. An analogous design was employed in the second experiment (n = 2) to desensitize the NKB receptor (neurokinin 3 receptor, NK3R) by administration of a continuous 48-hour i.v. infusion of senktide (200 μg/h). While a bolus of senktide during the last 3 h of continuous senktide administration failed to elicit GnRH release, thus confirming desensitization of NK3R, the ability of kisspeptin to stimulate GnRH was unimpaired. The foregoing findings support the view that NKB stimulation of GnRH release is upstream from KISS1R.

Fig. 1

Left-hand panel shows the LH response (mean plasma concentration ± SEM) to the last i.v. priming pulse of GnRH (G; gray arrow, 0.6 μg) and to a bolus injection of senktide (S; black arrow, 250 μg), administered 1 h later, on day 1 of the first experiment immediately before initiation of the continuous i.v. infusion of either kisspeptin-10 (100 μg/h, black data points) or vehicle (white data points) in agonadal juvenile male rhesus monkeys (n = 3). In the remaining panels, the effect of single sequential bolus injections of senktide, kisspeptin-10 (K; white arrow, 10 μg) or GnRH on LH during the last 4 h (shaded horizontal box) of the 99-hour continuous infusion of kisspeptin-10 or vehicle (day 4) is compared with the LH response to the same bolus injections of senktide and kisspeptin-10 administered 24 h (day 5) and 72 h (day 7) after termination of the respective continuous infusion. Note that the last data point describing the LH response to a given peptide challenge and that for the preinjection value of the subsequent challenge are the same. The greater LH discharge in response to the same senktide challenge on day 4 compared with that on day 1 is most likely due to the continued synthesis of LH after GnRH priming was terminated leading to a larger releasable pool of LH in the pituitary 4 days later. ∗ p ≤ 0.05, mean LH significantly different between continuous kisspeptin and vehicle infusion at time indicated.

Fig. 2

Effect of single bolus injections of GnRH (G; gray arrow, 0.6 μg), kisspeptin-10 (K; white arrow, 10 μg) or senktide (S; black arrow, 250 μg) on plasma LH concentrations prior to initiation (left-hand panels, day 1), during the last 4 h (shaded horizontal box; center panels, day 2) and after termination (right-hand panels, day 3) of the 48-hour continuous i.v. infusion of 200 μg/h of senktide (black data points) or vehicle (white data points) in 2 individual monkeys. Missing data points in bottom left-hand panel during the senktide challenge prior to initiation of the continuous infusion with this agonist are the result of sample loss during processing for storage. The senktide infusions were completed 5 days before initiation of the vehicle infusions. Note that the last data point describing the LH response to a given peptide challenge and that for the preinjection value of the subsequent challenge are the same. The greater LH discharge in response to the same kisspeptin challenge on day 4 compared with that on day 1 is most likely due to the continued synthesis of LH after GnRH priming was terminated leading to a larger releasable pool of LH in the pituitary 4 days later.