Discovery of potent kisspeptin antagonists delineate physiological mechanisms of gonadotropin regulation

Abstract

Neurons that produce gonadotropin-releasing hormone (GnRH) are the final common pathway by which the brain regulates reproduction. GnRH neurons are regulated by an afferent network of kisspeptin-producing neurons. Kisspeptin binds to its cognate receptor on GnRH neurons and stimulates their activity, which in turn provides an obligatory signal for GnRH secretion, thus gating down-stream events supporting reproduction. We have developed kisspeptin antagonists to facilitate the direct determination of the role of kisspeptin neurons in the neuroendocrine regulation of reproduction. In vitro and in vivo studies of analogues of kisspeptin-10 with amino substitutions have identified several potent and specific antagonists. A selected antagonist was shown to inhibit the firing of GnRH neurons in the brain of the mouse and to reduce pulsatile GnRH secretion in female pubertal monkeys; the later supporting a key role of kisspeptin in puberty onset. This analog also inhibited the kisspeptin-induced release of luteinizing hormone (LH) in rats and mice and blocked the postcastration rise in LH in sheep, rats, and mice, suggesting that kisspeptin neurons mediate the negative feedback effect of sex steroids on gonadotropin secretion in mammals. The development of kisspeptin antagonists provides a valuable tool for investigating the physiological and pathophysiological roles of kisspeptin in the regulation of reproduction and could offer a unique therapeutic agent for treating hormone-dependent disorders of reproduction, including precocious puberty, endometriosis, and metastatic prostate cancer.

Figure 1.

Peptide 234 is a potent inhibitor of kisspeptin-10 stimulation of IP. Substitution of Leu⁸ with d-Trp in combination with Ser⁵ substitution with Gly created potent antagonists (see supplemental Table S1, available at www.jneurosci.org as supplemental material). Additional substitution of Tyr¹ with d-Ala (234 shown here) enhanced this (*p < 0.05, **p < 0.01, ***p < 0.001). Peptide 234 alone had no intrinsic IP stimulation. Bars show mean ± SEM of five experiments.

Figure 2.

Peptide 234 antagonizes kisspeptin-10 excitation of GnRH neurons. Representative traces of GnRH neuronal firing rate over time. A, Increased GnRH firing rate after 1 nm kisspeptin-10 (bar). Downward spikes are individual action currents. B, Inhibition of kisspeptin-10 (1 nm) stimulation by peptide 234 (1 nm, bar). C, Summary bar graph showing mean ± SEM fold change in firing rate during baseline (white bars) and kisspeptin-10 (black bars); kisspeptin-10 significantly increased firing activity of GnRH neurons (n = 7; *p < 0.002). Response to kisspeptin-10 was significantly reduced with the presence of 1, 10, 100 nm peptide 234 (1 nm, n = 5; 10 nm, n = 6; 100 nm, n = 7; p < 0.001, all groups).

Figure 3.

Peptide 234 suppresses GnRH release in vivo. Representative cases from the effects of peptide 234 on GnRH release and group mean (±SEM; n = 6) are shown. A, Pulsatile GnRH release in the hypothalamus was suppressed by 10 nm peptide 234 infusion to the stalk-median eminence regions (dark shaded bar). Short arrows indicate GnRH peaks identified by PULSAR. B, In contrast, vehicle infusion as a control did not cause any significant changes in GnRH release (light shaded bar). C, Data analysis indicated that peptide 234 significantly (p < 0.05) suppressed GnRH release as compared with levels before peptide 234 as well as to the vehicle control. D, Vehicle infusion did not cause any significant changes. The estimated concentration of peptide 234 in the stalk-median eminence region was 1 nm, based on our previous assessment that the dialysis membrane passes ∼10% of peptides with a similar size. *p < 0.05 versus before peptide 234; ^ap < 0.05 versus control at corresponding time period.

Figure 4.

Peptide 234 effects on basal and kisspeptin-10-stimulated plasma LH in intact and castrated male rats. A, Peptide 234 inhibits kisspeptin-10-induced LH secretion in intact male rats. The animals were infused with 1 nmol peptide 234 at 0 and 60 min, followed by infusion of 100 pmol kisspeptin-10 with 1 nmol peptide 234 at 120 min. The peptide significantly inhibited LH production over the following 2 h (n = 10; *p < 0.05). B, Castrated male rats were given three infusions of 1 nmol peptide 234 at 0, 60, and 120 min, which significantly inhibited LH secretion after 240 min with 1 nmol peptide 234 (n = 10; p < 0.05). Bars show mean ± SEM.

Figure 5.

Peptide 234 inhibition of plasma LH in male mice. A, Two infusions of 15 nmol of peptide 234 inhibit the elevated LH levels of castrated male mice. B, Dose–response graph for castrated male mice given two infusions of peptide 234 indicates that all three doses tested are able to inhibit the postcastration rise in LH. C, Likewise, a single infusion of 5 or 15 nmol of peptide 234 potently inhibits LH levels of castrated mice. D, Exogenously administered kisspeptin (100 fmol) is unable to stimulate LH levels in intact male mice when 100 pmol peptide 234 is infused 5 min beforehand (n = 6–8). Bars show mean ± SEM (*p < 0.05).

Figure 6.

Central infusion of peptide 234 inhibits the secretory pulses of LH in OVX ewes. Concentrations of LH are shown in ewes treated with peptide 234 (closed bars) or control (opened bars). Arrows indicate LH pulses as defined in Materials and Methods. Analysis revealed a significant reduction in the mean LH concentration and pulse amplitude after peptide 234 infusion (see supplemental Table S2, available at www.jneurosci.org as supplemental material).