Kisspeptin, Neurokinin B, and Dynorphin Expression during Pubertal Development in Female Sheep

Abstract

The neural mechanisms underlying increases in gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion that drive puberty onset are unknown. Neurons coexpressing kisspeptin, neurokinin B (NKB), and dynorphin, i.e., KNDy neurons, are important as kisspeptin and NKB are stimulatory, and dynorphin inhibitory, to GnRH secretion. Given this, we hypothesized that kisspeptin and NKB expression would increase, but that dynorphin expression would decrease, with puberty. We collected blood and hypothalamic tissue from ovariectomized lambs implanted with estradiol at five, six, seven, eight (puberty), and ten months of age. Mean LH values and LH pulse frequency were the lowest at five to seven months, intermediate at eight months, and highest at ten months. Kisspeptin and NKB immunopositive cell numbers did not change with age. Numbers of cells expressing mRNA for kisspeptin, NKB, or dynorphin were similar at five, eight, and ten months of age. Age did not affect mRNA expression per cell for kisspeptin or NKB, but dynorphin mRNA expression per cell was elevated at ten months versus five months. Thus, neither KNDy protein nor mRNA expression changed in a predictable manner during pubertal development. These data raise the possibility that KNDy neurons, while critical, may await other inputs for the initiation of puberty.

Keywords: LH; dynorphin; kisspeptin; neurokinin B; puberty; sheep.

Figure 1

Diagram of changes in mean GnRH/LH secretion (solid line) and LH pulse frequency (lower panel insets) using a model wherein female sheep are OVX and implanted with a constant-release implant of E₂, indicated by the unchanging levels of E₂ with development (dotted line). During the prepubertal period (up to ~7 months), sensitivity to E₂-negative feedback is high and mean GnRH/LH secretion and LH pulse frequency is low. As animals grow and mature, sensitivity to E₂-negative feedback lessens, leading to an increase in GnRH/LH secretion and LH pulse frequency (~8 months). By ~9 months of age, the normal time of puberty, E₂-negative feedback has further lessened and GnRH/LH secretion and LH pulse frequency is maximal. The upper panel insets depict our hypothesis regarding changes in the KNDy neuron peptides kisspeptin (K), neurokinin B (N) and dynorphin (D). As kisspeptin and neurokinin B are stimulatory and dynorphin is inhibitory to GnRH/LH secretion, we hypothesize that expression of kisspeptin and neurokinin B will be minimal and that of dynorphin elevated during the prepubertal period in association with reduced GnRH/LH secretion. As the animal grows and matures, expression of the peptides will change in association with increased GnRH/LH secretion such that the expression of kisspeptin and neurokinin B will predominate and dynorphin will recede.

Figure 2

Mean LH (±SEM) (A), LH pulse frequency (±SEM) (B), and LH pulse amplitude (±SEM) (C) in OVX+E₂ lambs at 5, 6, 7, 8, and 10 months of age. Mean LH, LH pulse frequency, and LH pulse amplitude were lowest during the prepubertal ages of 5, 6, and 7 months, then increased to intermediate levels at the peripubertal time period of 8 months, and then were elevated above prepubertal values at the postpubertal age of 10 months. Differing letter superscripts denote significant (p < 0.05) differences. Panels (D–F) showing representative LH secretory profiles for individual lambs at 5, 8, and 10 months. Peaks of LH pulses are identified by asterisks. Number of animals: 5 months (n = 6), 6 months (n = 6), 7 months (n = 5), 8 months (n = 5), 10 months (n = 6). * p < 0.05.

Figure 3

Mean (±SEM) number of kisspeptin (black bars) and NKB (open bars) neurons (A) in female sheep at 5, 6, 7, 8, and 10 months of age as assessed by dual-label immunofluorescence. Mean (±SEM) percentage of kisspeptin neurons coexpressing NKB (black bars) and NKB neurons coexpressing kisspeptin (open bars) in those same lambs (B). Representative photomicrographs of kisspeptin (green), NKB (red), and merged (yellow) immunostaining in lambs at 5 months (C–E), 8 months (F–H), and 10 months (I–K) of age. Scale bars = 200 µm. Number of animals: 5 months (n = 6), 6 months (n = 6), 7 months (n = 5), 8 months (n = 5), 10 months (n = 6).

Figure 4

Kisspeptin mRNA (Kiss1) in the arcuate nucleus of female sheep at different ages. Low-power confocal images (20× objective) from a 5- (A), 8- (B), and 10-month-old female (C). Insets are a single-plane representative cell taken at high-power (63× objective) within the respective section. (D) Mean (±SEM) number of cells expressing mRNA for kisspeptin. (E) Mean (±SEM) integrated density per cell. Asterisk denotes a tendency (p = 0.065) for the values at month 10 to be greater than those at month 5. Scale bar, 50 µm. Inset scale bar, 10 µm. Cells expressing mRNA for kisspeptin were artificially colored green. Number of animals: 5 months (n = 6), 8 months (n = 5), 10 months (n = 6).

Figure 5

NKB mRNA (TAC3) in the arcuate nucleus of female sheep at different ages. Low-power confocal images (20× objective) from a 5- (A), 8- (B), and 10-month (C) female. Insets are a single-plane representative cell taken at high-power (63× objective) within the respective section. (D) Mean (±SEM) number of cells expressing mRNA for NKB mRNA. (E) Mean (±SEM) integrated density per cell. Scale bar, 50 µm. Inset scale bar, 10 µm. Cells expressing mRNA for NKB were artificially colored blue. Number of animals: 5 months (n = 6), 8 months (n = 5), 10 months (n = 6).

Figure 6

Dynorphin mRNA (PDyn) in the arcuate nucleus of female sheep at different ages. Low-power confocal images (20× objective) from a 5- (A), 8- (B), and 10-month (C) female. Insets are a single-plane representative cell taken at high-power (63× objective) within the respective section. (D) Mean (±SEM) number of cells expressing mRNA for dynorphin. (E) Mean (±SEM) integrated density per cell. Differing letter superscripts denote significant (p < 0.05) differences. Scale bar, 50 µm. Inset scale bar, 10 µm. Cells expressing mRNA for dynorphin were artificially colored red. Number of animals: 5 months (n = 6), 8 months (n = 5), 10 months (n = 6).