The distribution of the cells expressing three prepro-gonadotrophin-releasing hormones (GnRH), corresponding to salmon GnRH (sGnRH), seabream GnRH (sbGnRH), and chicken GnRH-II (cGnRH-II) forms, was studied in the brain and pituitary of the sea bass (Dicentrarchus labrax) by using immunohistochemistry. To circumvent the cross-reactivity problems of antibodies raised to GnRH decapeptides, we used specific antibodies generated against the different sea bass GnRH-associated peptides (GAP): salmon GAP (sGAP), seabream GAP (sbGAP), and chicken-II GAP (cIIGAP). The salmon GAP immunostaining was mostly detected in terminal nerve neurons but also in ventral telencephalic and preoptic perikarya. Salmon GAP-immunoreactive (ir) fibers were observed mainly in the forebrain, although sGAP-ir projections were also evident in the optic tectum, mesencephalic tegmentum, and ventral rhombencephalon. The pituitary only receives a few sGAP-ir fibers. The seabream GAP-ir cells were mainly detected in the preoptic area. Nevertheless, sbGAP-ir neurons were also found in olfactory bulbs, ventral telencephalon, and ventrolateral hypothalamus. The sbGAP-ir fibers were only observed in the ventral forebrain, innervating strongly the pituitary gland. Finally, chicken-II GAP immunoreactivity was only detected in large synencephalic cells, which are the origin of a profuse innervation reaching the telencephalon, preoptic area, hypothalamus, thalamus, pretectum, posterior tuberculum, mesencephalic tectum and tegmentum, cerebellum, and rhombencephalon. However, no cIIGAP-ir fibers were detected in the hypophysis. These results corroborate the overlapping of sGAP- and sbGAP-expressing cells in the forebrain of the sea bass, and provide, for the first time, unambiguous information on the distribution of projections of the three different GnRH forms expressed in the brain of a single species.

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Kisspeptin is an important regulator of reproduction in many vertebrates. The involvement of the two kisspeptins, Kiss1 and Kiss2, and their receptors, Gpr54-1 and Gpr54-2, in controlling reproduction was studied in the brains of the modern teleosts, striped and hybrid basses. In situ hybridization and laser capture microdissection followed by quantitative RT (QRT)-PCR detected coexpression of kiss1 and kiss2 in the hypothalamic nucleus of the lateral recess. Neurons expressing gpr54-1 and gpr54-2 were detected in several brain regions. In the preoptic area, gpr54-2 was colocalized in GnRH1 neurons while gpr54-1 was expressed in cells attached to GnRH1 fibers, indicating two different modes of GnRH1 regulation. The expression of all four genes was measured in the brains of males and females at different life stages using QRT-PCR. The levels of kiss1 and gpr54-1 mRNA, the latter being expressed in minute levels, were consistently lower than those of kiss2 and gpr54-2. While neither gene's expression increased at prepuberty, all were dramatically elevated in mature females. The levels of kiss2 mRNA increased also in mature males. Kiss1 peptide was less potent than Kiss2 in elevating plasma luteinizing hormone levels and in up-regulating gnrh1 and gpr54-2 expression in prepubertal hybrid bass in vivo. In contrast, during recrudescence, Kiss1 was more potent than Kiss2 in inducing luteinizing hormone release, and Kiss2 down-regulated gnrh1 and gpr54-2 expression. This is the first report in fish to demonstrate the alternating actions and the importance of both neuropeptides for reproduction. The organization of the kisspeptin system suggests a transitional evolutionary state between early to late evolving vertebrates.

In recent years, crustacean hyperglycemic hormones (CHHs), traditionally identified in the medulla terminalis X-organ and sinus gland (SG) in the eyestalk (ES), have been found in non-ES tissues. Reported sites for the synthesis of CHH-like neuropeptides in non-ES tissues include the gut [1,2], subesophageal ganglion (SOG) [3], pericardial organs (POs) [4,5], and cells in the abdominal segments of embryos [6]. In Carcinus maenas, the expression and translation of gut CHH occurs exclusively during premolt [1,2], whereas ES-CHH is molt stage independent [7]. The amino acid sequence of gut CHH is identical to that in the ES [1,8], but only 66% homologous to 5,8]. Thus, it seems that C. maenas [1,4,8] The rapid increase in the number of putative cDNA sequences encoding crustacean hyperglycemic hormone (CHH) family in various tissues [either from the eyestalk (ES) or elsewhere] underscores a need to identify the corresponding neuropeptides in relevant tissues. Moreover, the presence of provided structural CHH implies the level of the complexity of physiological regulation in crustaceans. Much less is known of the functions of non-ES CHH than of those of its counterpart present in ESs. In the blue crab, Callinectes sapidus, we know little of CHH involvement in response to the stressful conditions that naturally occur in Chesapeake Bay. We have identified two isoforms of CHH neuropeptide in the sinus gland of the ES and isolated a full-length cDNA encoding CHH from the pericardial organ (PO). The functions of ES-CHH and PO-CHH in this species were studied with regard to expression and release in response to stressful episodes: hypoxia, emersion, and temperatures. Animals exposed to hypoxic conditions responded with concomitant release of both CHHs. In contrast, the mRNA transcripts encoding two CHHs were differentially regulated: PO-CHH increased, whereas ES-CHH decreased. This result suggests a possible differential regulation of transcription of these CHHs.