Interactions between kisspeptin and neurokinin B in the control of GnRH secretion in the female rat

Abstract

Neurokinin B (NKB) and its cognate receptor neurokinin 3 (NK3R) play a critical role in reproduction. NKB and NK3R are coexpressed with dynorphin (Dyn) and kisspeptin (Kiss1) genes in neurons of the arcuate nucleus (Arc). However, the mechanisms of action of NKB as a cotransmitter with kisspeptin and dynorphin remain poorly understood. We explored the role of NKB in the control of LH secretion in the female rat as follows. 1) We examined the effect of an NKB agonist (senktide, 600 pmol, administered into the lateral cerebral ventricle) on luteinizing hormone (LH) secretion. In the presence of physiological levels of estradiol (E(2)), senktide induced a profound increase in serum levels of LH and a 10-fold increase in the number of Kiss1 neurons expressing c-fos in the Arc (P < 0.01 for both). 2) We mapped the distribution of NKB and NK3R mRNAs in the central forebrain and found that both are widely expressed, with intense expression in several hypothalamic nuclei that control reproduction, including the Arc. 3) We studied the effect of E(2) on the expression of NKB and NK3R mRNAs in the Arc and found that E(2) inhibits the expression of both genes (P < 0.01) and that the expression of NKB and NK3R reaches its nadir on the afternoon of proestrus (when circulating levels of E(2) are high). These observations suggest that NKB/NK3R signaling in Kiss1/NKB/Dyn-producing neurons in the Arc has a pivotal role in the control of gonadotropin-releasing hormone (GnRH)/LH secretion and its regulation by E(2)-dependent negative feedback in the rat.

Fig. 1.

Effects of acute administration of senktide on luteinizing hormone (LH) release in intact female rats at diestrus 1 (A) and proestrus (B). Senktide (600 pmol) or vehicle was acutely injected intracerebroventricularly. Blood samples for LH determination were obtained before [0 min (0′)] and 20, 60, and 120 min after injection. In addition to time course profiles, integrated secretory responses to central administration of senktide [calculated as area under the curve (AUC) over the 120-min study period] are shown. Hormone values are means ± SE. Statistical significance was determined by 2-way ANOVA: *P < 0.01 vs. corresponding control values; ^aP < 0.01 vs. senktide basal (0′); ^bP < 0.05 vs. senktide basal.

Fig. 2.

Effects of acute administration of senktide on LH release in ovariectomized (OVX) rats implanted subcutaneously with a sham (oil) capsule (A) and with an estradiol (E₂)-filled capsule (OVX + E₂, B). Senktide (600 pmol) or vehicle was acutely injected intracerebroventricularly. Blood samples for LH determination were obtained before (0 min) and 20, 60, and 120 min after injection. In addition to time course profiles, integrated secretory responses to central administration of senktide (calculated as AUC over the 120-min study period) are shown. Hormone values are means ± SE. *P < 0.01 vs. corresponding control values; ^aP < 0.01 vs. senktide basal (2-way ANOVA).

Fig. 3.

A and B: photomicrographs of kisspeptin 1 (Kiss1) and c-fos mRNA in situ hybridization in OVX + E₂ rats 30 min after vehicle or senktide treatment. Red stain indicates cells expressing Kiss1 mRNA; white grains indicate cells expressing c-fos mRNA. Scale bars, 25 μm. C: percentage of Kiss1 cells expressing c-fos mRNA in the arcuate nucleus (Arc). Values are means ± SE. *P < 0.01 (Student's t-test).

Fig. 4.

Schematic representation of distribution of neurokinin B (NKB) mRNA (top) and NKB receptor (NK3R) mRNA (bottom) in brain of adult female rat. No differences in general distribution of transcripts were observed between treatments (OVX + sham vs. OVX + E₂). Both transcripts were widely distributed throughout the brain but concentrated in discrete areas. BST, bed nucleus of the stria terminalis; COAa, anterocortical amygdaloid nucleus; SON, supraoptic nucleus; BLAa, basolateral nucleus of amygdala; MH, medial habenular nucleus; LHA, lateral hypothalamus; ZI, zona incerta; SCN, suprachiasmatic nucleus; VMH, ventromedial hypothalamic nucleus; PVH, paraventricular hypothalamic nucleus.

Fig. 5.

Total NKB mRNA in the Arc of adult female rats (no. of cells × grains per cell). A: total mRNA in intact cycling females in different phases of estrous cycle [morning of diestrus (D1 AM), morning of proestrus (P AM), and afternoon of proestrus (P PM)]. B: total mRNA in OVX + sham or OVX + E₂ rats. Values are means ± SE. *P < 0.01 (1-way ANOVA). C: representative photomicrographs of NKB mRNA in situ hybridization in different phases of the estrous cycle. 3V, 3rd ventricle. Scale bars, 50 μm.

Fig. 6.

Total NK3R mRNA in the Arc of adult female rats (no. of cells × grains per cell). Left: total mRNA in intact cycling females in different phases of the estrous cycle. Right: total mRNA in OVX + sham or OVX + E₂ rats. Values are means ± SE. *P < 0.01 (1-way ANOVA). #P < 0.01 (Student's t-test).