Kisspeptins are new actors in the neuroendocrine regulation of reproduction. In vertebrates, the number of kiss genes varies from none to three. Zebrafish have two kiss genes, kiss1 and kiss2, and two kiss receptors (GPR54), kiss1r and kiss2r. To provide detailed information on the organization of the kiss systems in zebrafish, antibodies were raised against the C terminus of zebrafish preproKiss1 and preproKiss2. Immunohistochemistry fully confirmed in situ hybridization data, showing that kiss1-expressing neurons are only located in the habenular nucleus, while kiss2-expressing neurons are found in the dorsal and ventral hypothalamus. Kiss1-expressing cells project only to the interpeduncular and raphe nuclei and strongly expressed the kiss1r receptor. In contrast, kiss2-expressing cells are mostly present in the dorsal and ventral hypothalamus and project widely into the subpallium, the preoptic area, the thalamus, the ventral and caudal hypothalamus, and the mesencephalon. All these regions strongly expressed the kiss2r messengers. Kiss2 fibers profusely innervate the ventral forebrain and notably made close apposition with GnRH3 neurons. Estrogen treatment of juvenile fish with estradiol causes increase in kiss2 and kiss2r expression. In the pituitary gland, no proKiss2- positive fibers were detected, while positive cells were observed in the pars intermedia. In addition to proposing a successful strategy to develop antibodies to kisspeptins, these data indicate that the kiss2 systems of zebrafish are implicated in reproductive events, while the kiss1 gene would play other functions that remain to be established.

This study, conducted in the brain of a perciform fish, the European sea bass, aimed at raising antibodies against the precursor of the kisspeptins in order to map the kiss systems and to correlate the expression of kisspeptins, kiss1 and kiss2, with that of kisspeptin receptors (kiss-R1 and kiss-R2). Specific antibodies could be raised against the preprokiss2, but not the preoprokiss1. The data indicate that kiss2 neurons are mainly located in the hypothalamus and project widely to the subpallium and pallium, the preoptic region, the thalamus, the pretectal area, the optic tectum, the torus semicircularis, the mediobasal medial and caudal hypothalamus, and the neurohypophysis. These results were compared to the expression of kiss-R1 and kiss-R2 messengers, indicating a very good correlation between the wide distribution of Kiss2-positive fibers and that of kiss-R2 expressing cells. The expression of kiss-R1 messengers was more limited to the habenula, the ventral telencephalon and the proximal pars distalis of the pituitary. Attempts to characterize the phenotype of the numerous cells expressing kiss-R2 showed that neurons expressing tyrosine hydroxylase, neuropeptide Y and neuronal nitric oxide synthase are targets for kisspeptins, while GnRH1 neurons did not appear to express kiss-R1 or kiss-R2 messengers. In addition, a striking result was that all somatostatin-positive neurons expressed-kissR2. These data show that kisspeptins are likely to regulate a wide range of neuronal systems in the brain of teleosts.