Surge-Like Luteinising Hormone Secretion Induced by Retrochiasmatic Area NK3R Activation is Mediated Primarily by Arcuate Kisspeptin Neurones in the Ewe

Abstract

The neuropeptides neurokinin B (NKB) and kisspeptin are potent stimulators of gonadotrophin-releasing hormone (GnRH)/luteinsing hormone (LH) secretion and are essential for human fertility. We have recently demonstrated that selective activation of NKB receptors (NK3R) within the retrochiasmatic area (RCh) and the preoptic area (POA) triggers surge-like LH secretion in ovary-intact ewes, whereas blockade of RCh NK3R suppresses oestradiol-induced LH surges in ovariectomised ewes. Although these data suggest that NKB signalling within these regions of the hypothalamus mediates the positive-feedback effects of oestradiol on LH secretion, the pathway through which it stimulates GnRH/LH secretion remains unclear. We proposed that the action of NKB on RCh neurones drives the LH surge by stimulating kisspeptin-induced GnRH secretion. To test this hypothesis, we quantified the activation of the preoptic/hypothalamic populations of kisspeptin neurones in response to POA or RCh administration of senktide by dual-label immunohistochemical detection of kisspeptin and c-Fos (i.e. marker of neuronal activation). We then administered the NK3R agonist, senktide, into the RCh of ewes in the follicular phase of the oestrous cycle and conducted frequent blood sampling during intracerebroventricular infusion of the kisspeptin receptor antagonist Kp-271 or saline. Our results show that the surge-like secretion of LH induced by RCh senktide administration coincided with a dramatic increase in c-Fos expression within arcuate nucleus (ARC) kisspeptin neurones, and was completely blocked by Kp-271 infusion. We substantiate these data with evidence of direct projections of RCh neurones to ARC kisspeptin neurones. Thus, NKB-responsive neurones in the RCh act to stimulate GnRH secretion by inducing kisspeptin release from KNDy neurones.

Keywords: KNDy neurones; LH surge; NK3R; kisspeptin; retrochiasmatic area; tachykinins.

Fig. 1

Effect of retrochiasmatic area (RCh) senktide administration on kisspeptin cell activation in anoestrous ewes. (A) Mean ± SEM luteinising hormone (LH) secretion in anoestrous ewes during the first 3 h after receiving bilateral senktide-containing (n = 7) or empty (n = 5) implants in the RCh. (B) Percentage (mean ± SEM) of arcuate nucleus (ARC) or preoptic area (POA) kisspeptin neurones coexpressing c-Fos in those two treatment groups. *P < 0.05 versus corresponding value in control group (Mann–Whitney rank sum test).

Fig. 2

Effect of preoptic area (POA) senktide administration on kisspeptin cell activation in follicular phase ewes. (A) Mean ± SEM luteinising hormone (LH) secretion in follicular phase ewes during the first 3 h after receiving bilateral intra-POA senktide-containing (n = 6) or empty (n = 5) implants. (B) Percentage (mean ± SEM) of arcuate nucleus (ARC) or POA kisspeptin neurones coexpressing c-Fos in those two treatment groups. *P < 0.05 versus corresponding value in control group (Mann–Whitney rank sum test). Note that the scale of the y-axis is the same as in Fig. 1 to facilitate comparison of the magnitudes of the response to senktide in both areas.

Fig. 3

Retrochiasmatic area (RCh) biotinylated dextran amine injection sites in three ewes. (A) Ewe #602: rostral–caudal spread of approximately 1.2 mm (B). Ewe #913: rostral–caudal spread of approximately 0.5 mm. (C) Ewe #48: rostral–caudal spread of approximately 1.5 mm. Dotted lines indicate the approximate boundaries of the RCh based on matched Nissl-stained sections at the same level. Scale bar = 500 μm. 3V, third ventricle; ot, optic tract.

Fig. 4

Arcuate nucleus (ARC) projections of retrochiasmatic area neurones in three ewes. Demonstration of biotinylated dextran amine (BDA)-labelled axons in ARC in close proximity to kisspeptin immunoreactive cell bodies in ewes #48 (A–C), #602 (D–F), and #914 (G–I). (A, D, G) show low magnification overview of labelling in ARC; scale bar = 50 μm. (B, C, E, F, H, I) show higher magnifications of areas indicated by boxes in (A, D, G) and illustrate BDA-labelled boutons in close proximity to kisspeptin neurones (indicated by arrows); scale bar = 15 μm.

Fig. 5

Close contacts between retrochiasmatic area neurones and arcuate nucleus kisspeptin neurones (indicated by arrows). Confocal images (optical sections of 1 μm) illustrating biotinylated dextran amine (BDA)-labelled axon terminals (red) in close apposition to kisspeptin-immunoreactive soma and dendrites (green) in ewes #602 (A) and #48 (B, C). Retrogradely-labelled cells and dendrites containing BDA (arrow head) are shown in (C). Note that this neurone is negative for kisspeptin immunoreactivity. Scale bar = 15 μm.

Fig. 6

Effect of Kiss1R antagonist on retrochiasmatic area (RCh) senktide-induced luteinising hormone (LH) secretion in follicular phase ewes. (A) Camera lucida representation of ovine hypothalamus showing correct placement of bilateral guide cannulae within the RCh in seven animals. Dashed lines connect bilateral microimplant loci in the same animal. 3V, third ventricle; PVN, paraventricular nucleus; fx, fornix; ot, optic tract. (B) LH secretion, expressed as the area under the curve [mean ± SEM area under the curve (AUC)] in ovary-intact ewes (n = 7 per treatment group) receiving senktide implants in the RCh during i.c.v. infusion of the Kiss1R antagonist, Kp-271 (black bar) or vehicle (white bar) during the follicular phase of the oestrous cycle. (C, D) Representative LH profiles demonstrating the effect of intra-RCh implantation (white bar) of senktide, during i.c.v. infusion (black bar) of the Kiss1R antagonist, Kp-271, or vehicle (saline), on LH secretion in ovary-intact ewes during the follicular phase of the oestrous cycle. (E) Concentrations of LH (mean ± SEM) in ovary-intact ewes (n = 7 per treatment group) bilaterally implanted with senktide in the RCh (white bar) during i.c.v. infusion (black bar) of the Kiss1R antagonist, Kp-271 (black triangles), or vehicle (saline; white circles) during the follicular phase of the oestrous cycle. *P < 0.05 versus corresponding value in saline-treated control group (Wilcoxon signed rank test); #first value in the time-series that is significantly different (P < 0.05 versus corresponding value in saline-treated control group [two-way ANOVA with repeated measures (influence of time and treatment) with Holm–Sidak pairwise multiple comparison], all subsequent values are also significantly different from controls).

Fig. 7

Schematic representation of connectivity between neurones implicated in the luteinising hormone surge in sheep. Retrochiasmatic area (RCh) neurones (grey) project to KNDy neurones in the arcuate nucleus (ARC), which coexpress kisspeptin (purple), NKB (blue) and dynorphin (Dyn) (brown), and innervate kisspeptin and gonadotrophin-releasing hormone neurones (GnRH) (green) in the preoptic area (POA). The source of NKB that activates NK3R on RCh neurones is currently unknown. ac, anterior commissure; inf, infundibular recess; OCh, optic chiasm.