Figure 1.
Luciferase assays for the activation of two types of receptors, Gpr54-1 and Gpr54-2, by the ligands, Kiss1 and Kiss2.
Medaka gpr54-1 (A, C) or gpr54-2 (B, D) cDNA was transfected to COS-7 cells with SRE-luc or CRE-luc vector. Various concentrations of medaka Kiss1 and Kiss2 were applied to the culture medium, and the luciferase activity was measured. The results are indicated as mean ± SEM, each of which was conducted in triplicates. The data are expressed as the ratio of changes in luciferase activity over the control renilla luciferase activity.
Figure 2.
DIG-labelled in situ hybridization of gpr54-1 shows localization of gpr54-1 mRNA positive cells.
gpr54-1 mRNA positive neurons are localized in POp, POm (A), Vd/Vs/Vp (not shown), and habenula (Hb; B). Scale bars: 50 µm.
Figure 3.
DIG-labelled in situ hybridization of gpr54-2 shows localization of gpr54-2 mRNA positive cells.
gpr54-2 mRNA positive neurons are localized in the boundary between telencephalon (Tel) and olfactory bulb (OB; A), area ventralis telencephali pars dorsalis/supracommissuralis/posterior (Vd;B/Vs;C/Vp;D), area preoptica (POA; E), nucleus preopticus pars magnocellularis (POm; F), nucleus diffusus tori lateralis (NDTL; G), nucleus posterioris periventricularis (NPPv; H), nucleus ventralis tuberis (NVT; I), nucleus recessus lateralis (NRL; J), and corpus mammillare (CM, not shown). Scale bars: 50 µm.
Figure 4.
Schematic illustration of the distribution of kisspeptin receptors in medaka brain.
gpr54-1- and gpr54-2-expressing neurons are mainly localized in the ventral telencephalon, preoptic area, and hypothalamus, suggesting their functions in homeostatic and behavioral regulations. Note that gpr54-1 is also expressed in habenula but not in NIP, which is innervated by habenular neurons, suggesting the autocrine/paracrine regulation of habenular neurons by kisspeptins. Tel, telencephalon; TeO, optic tectum; ca, anterior commissure; Cb, cerebellum; Hy, hypothalamus; Hb, habenula; fr, fasciculus retroflexus; NIP, interpeduncular nucleus.
Figure 5.
Dual fluorescence in situ hybridization showing that isotocin and vasotocin neurons express gpr54-2.
Isotocin neurons (A; green) and vasotocin neurons (D; green) express gpr54-2 (B, E; magenta). Merged photographs are shown in C and F. On the other hand, neurons that do not express isotocin or vasotocin (green) express gpr54-1(magenta) mRNA (G–I, and J–L, respectively). Scale bar: 50 µm.
Figure 6.
DIG-labelled in situ hybridization of the brain regions surrounding the three types of GnRH neurons (B: gnrh1, E: gnrh2, H: gnrh3), showing the presence or absence of expressions for the kisspeptin receptors, gpr54-1 or gpr54-2, in these regions.
The results for gpr54-1 mRNA signals (A, D, G) and gpr54-2 mRNA signals (C, F, I) show that the gpr54-1- and gpr54-2-expressing neurons are localized in the POA surrounding the GnRH1 neurons (A–C), but not in the midbrain tegmentum surrounding the GnRH2 neurons (D–F) or the ventral telencephalic area surrounding the GnRH3 neurons (G–I). Scale bars: 50 µm.
Figure 7.
Dual fluorescence in situ hybridization showing that kisspeptin receptors (gpr54-1 and -2) are highly expressed in the neurons (magenta) adjacent to the GnRH1 neurons (green) in the POA.
gpr54-1 mRNA (B) is expressed by neurons in proximity to the ventrolateral group of GnRH1 neurons in the POA (A; merged photo in C). gpr54-2 mRNA (E) is expressed in neurons near the dorsomedial group of GnRH1 neurons in the POA (D; merged photo in F). Scale bar: 50 µm.