Even though for almost years GnRH was considered the main

Even though for almost 30 years GnRH was considered the main neuropeptide regulating gonadotropin release in vertebrates, Tsutsui et al. (2000) discovered, in the Japanese quail, Coturnix japonica, a hypothalamic neuropeptide that directly inhibits gonadotropin release, and thus named as gonadotropin-inhibitory hormone (GnIH). GnIH is a member of the RFamide peptide family that includes among others, kisspeptin and neuropeptide FF (NPFF). In the last years, GnIH orthologs have been identified in tetrapods, fish and prothocordates (Sawada et al., 2002, Ubuka et al., 2009, Osugi et al., 2015, Muñoz-Cueto et al., 2017, Tsutsui et al., 2017). However, in fish, the function of GnIH as an inhibitory factor of reproduction is far from being understood (Muñoz-Cueto et al., 2017); for this reason, some authors prefer to name it as LPXRFamide peptide. The location of GnIH neurons is variable among different vertebrate species, although the presence of a population of GnIH neurons in the periventricular region of the preoptic/hypothalamic area and a profuse innervation of almost all plx4032 regions are common characteristics of all studied vertebrates (Muñoz-Cueto et al., 2017). In avians and mammals, an interaction between GnIH and GnRH neurons was demonstrated, as well as GnIH fiber projections to the median eminence, evidencing that this peptide may affect the reproductive axis either directly on gonadotrops or indirectly via GnRH neurons (Kriegsfeld et al., 2006, Ubuka et al., 2008, Tsutsui and Ubuka, 2016, Peragine et al., 2017). In fish, pituitary innervation by GnIH fibers has not been demonstrated to be a general characteristic, and different scenarios have been observed with respect to the relationship between GnIH and GnRH neurons. While in zebrafish GnIH fibers reach the pituitary, and contact GnRH3 neurons, the hypophysiotropic variant in this species (Spicer et al., 2017), in tilapia, GnIH fibers also reach the pituitary, but no association was observed between GnIH fibers and GnRH1 nor GnRH3 cells (Ogawa et al., 2016). In our experimental species, the South American cichlid fish Cichlasoma dimerus, three GnRHs variants have been identified: GnRH1 (sbGnRH), GnRH2 (cGnRH2) and GnRH3 (sGnRH). The distribution pattern was previously determined, showing that neurons expressing GnRH1 are mainly localized in the preoptic area (POA), while GnRH3 mostly in the forebrain, but some overlapping pattern is observed. These two variants were shown to innervate the pituitary gland, being GnRH1 the most prominent on this respect. As in any other bony fish, GnRH2 expressing cells, are located in the midbrain and their fibers are mainly distributed along the mid and hindbrain but they do not innervate the pituitary gland (Pandolfi et al., 2005). GnIH neurons are located in two discrete nuclei: the nucleus posterioris periventricularis (NPP) of the hypothalamus and in the nucleus olfacto-retinalis (NOR). GnIH-immunoreactive fibers are present in all brain regions, presenting high density in the nucleus lateralis tuberis (NLT) at both sides of the third ventricle, but no fibers were observed at the pituitary level in adult fish (Di Yorio et al., 2016). Taking into account the scarce information on the relationship between GnIH and GnRH neurons in fish, and considering that morphological associations allow us to infer physiological interactions between these systems, the aim of the present study was to evaluate the anatomical relationship between neurons expressing GnIH and the three GnRH variants in Cichlasoma dimerus.
Materials and methods
Results Taking into account the distribution of GnRHs and GnIH neurons previously described in Pandolfi et al., 2005, Di Yorio et al., 2016, the analysis was focused in those areas were somas and a high density of fibers were observed (Fig. 1).
Discussion In the present work, the anatomical relationship between neurons and fibers expressing GnIH and GnRH variants were studied in the cichlid fish, C. dimerus. This study showed that GnIH-ir neurons co-expressed GnRH3 in the NOR and GnIH-ir fibers presented contacts with GnRH2-ir fibers but not with GnRH1-ir perikarya and cell processes.