Sexual Dimorphic Distribution of Hypothalamic Tachykinin1 Cells and Their Innervations to GnRH Neurons in the Zebrafish.

Substance P (SP) and neurokinin A (NKA), encoded by TAC1/Tac1 gene are members of the tachykinin family, which exert their neuromodulatory roles in vertebrate reproduction. In mammals, SP and NKA have been shown to regulate gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion via kisspeptin neurons. On the other hand, the role of SP/NKA in the regulation of reproduction in non-mammalian vertebrates is not well known. In the present study, we first localized expression of tac1 mRNA in the brain of male and female zebrafish, Danio rerio. Next, using an antibody against zebrafish tachykinin1 (Tac1), we examined the neural association of SP/NKA neural processes with GnRH3 neurons, and with kisspeptin (kiss2) neurons, in the brains of male and female zebrafish. In situ hybridization showed an apparent male-dominant tac1 expression in the ventral telencephalic area, the anterior and posterior parts of the parvocellular preoptic nucleus, and the suprachiasmatic nucleus. On the other hand, there was female-dominant tac1 expression in the ventral periventricular hypothalamus. Confocal images of double-labeled zebrafish Tac1 and GnRH3 showed associations between Tac1-immunoreactive processes and GnRH3 neurons in the ventral telencephalic area. In contrast, there was no apparent proximity of Tac1 processes to kiss2 mRNA-expressing neurons in the hypothalamus. Lastly, to elucidate possible direct action of SP/NKA on GnRH3 or Kiss2 neurons, expression of SP/NKA receptor, tacr1a mRNA was examined in regions containing GnRH3 or Kiss2 neurons by in situ hybridization. Expression of tacr1a mRNA was seen in several brain regions including the olfactory bulb, preoptic area and hypothalamus, where GnRH3 and Kiss2 cells are present. These results suggest that unlike in mammals, Tac1 may be involved in male reproductive functions via direct action on GnRH3 neurons but independent of kisspeptin in the zebrafish.

Distribution of Kiss2 receptor in the brain and its localization in neuroendocrine cells in the zebrafish.

Kisspeptin is a hypothalamic neuropeptide, which acts directly on gonadotropin-releasing hormone (GnRH)-secreting neurons via its cognate receptor (GPR54 or Kiss-R) to stimulate GnRH secretion in mammals. In non-mammalian vertebrates, there are multiple kisspeptins (Kiss1 and Kiss2) and Kiss-R types. Recent gene knockout studies have demonstrated that fish kisspeptin systems are not essential in the regulation of reproduction. Studying the detailed distribution of kisspeptin receptor in the brain and pituitary is important for understanding the multiple action sites and potential functions of the kisspeptin system. In the present study, we generated a specific antibody against zebrafish Kiss2-R (=Kiss1Ra/GPR54-1/Kiss-R2/KissR3) and examined its distribution in the brain and pituitary. Kiss2-R-immunoreactive cell bodies are widely distributed in the brain including in the dorsal telencephalon, preoptic area, hypothalamus, optic tectum, and in the hindbrain regions. Double-labeling showed that not all but a subset of preoptic GnRH3 neurons expresses Kiss2-R, while Kiss2-R is expressed in most of the olfactory GnRH3 neurons. In the posterior preoptic region, Kiss2-R immunoreactivity was seen in vasotocin cells. In the pituitary, Kiss2-R immunoreactivity was seen in corticotropes, but not in gonadotropes. The results in this study suggest that Kiss2 and Kiss2-R signaling directly serve non-reproductive functions and indirectly subserve reproductive functions in teleosts.

Distribution of LPXRFa, a gonadotropin-inhibitory hormone ortholog peptide, and LPXRFa receptor in the brain and pituitary of the tilapia.

In vertebrates, gonadotropin-releasing hormone (GnRH) and gonadotropin-inhibitory hormone (GnIH), respectively, regulate reproduction in positive and negative manners. GnIH belongs to the LPXRFa family of peptides previously identified in mammalian and nonmammalian vertebrates. Studying the detailed distribution of LPXRFa as well as its receptor (LPXRFa-R) in the brain and pituitary is important for understanding their multiple action sites and potential functions. However, the distribution of LPXRFa and LPXRFa-R has not been studied in teleost species, partially because of the lack of fish-specific antibodies. Therefore, in the present study, we generated specific antibodies against LPXRFa and its receptor from Nile tilapia (Oreochromis niloticus), and examined their distributions in the brain and pituitary by immunohistochemistry. Tilapia LPXRFa-immunoreactive neurons lie in the posterior ventricular nucleus of the caudal preoptic area, whereas LPXRFa-R-immunoreactive cells are distributed widely. Double immunofluorescence showed that neither LPXRFa-immunoreactive fibers nor LPXRFa-R is closely associated or coexpressed with GnRH1, GnRH3, or kisspeptin (Kiss2) neurons. In the pituitary, LPXRFa fibers are closely associated with gonadotropic endocrine cells [expressing luteinizing hormone (LH) and follicle-stimulating hormone (FSH)], with adrenocorticomelanotropic cells [corticotropin (ACTH) and α-melanotropin (α-MSH)], and with somatolactin endocrine cells. In contrast, LPXRFa-R are expressed only in LH, ACTH, and α-MSH cells. These results suggest that LPXRFa and LPXRFa-R signaling acts directly on the pituitary cells independent from GnRH or kisspeptin and could play multiple roles in reproductive and nonreproductive functions in teleosts. J. Comp. Neurol. 524:2753-2775, 2016. © 2016 Wiley Periodicals, Inc.