Novel Pituitary Actions of TAC4 Gene Products in Teleost.

Tachykinin 4 (TAC4) is the latest member of the tachykinin family involved in several physiological functions in mammals. However, little information is available about TAC4 in teleost. In the present study, we firstly isolated TAC4 and six neurokinin receptors (NKRs) from grass carp brain and pituitary. Sequence analysis showed that grass carp TAC4 could encode two mature peptides (namely hemokinin 1 (HK1) and hemokinin 2 (HK2)), in which HK2 retained the typical FXGLM motif in C-terminal of tachyinin, while HK1 contained a mutant VFGLM motif. The ligand-receptor selectivity showed that HK2 could activate all 6 NKRs but with the highest activity for the neurokinin receptor 2 (NK2R). Interestingly, HK1 displayed a very weak activation for each NKR isoform. In grass carp pituitary cells, HK2 could induce prolactin (PRL), somatolactin α (SLα), urotensin 1 (UTS1), neuromedin-B 1 (NMB1), cocaine- and amphetamine-regulated transcript 2 (CART2) mRNA expression mediated by NK2R and neurokinin receptor 3 (NK3R) via activation cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA), phospholipase C (PLC)/inositol 1,4,5-triphosphate (IP3)/protein kinase C (PKC) and calcium2+ (Ca2+)/calmodulin (CaM)/calmodulin kinase-II (CaMK II) cascades. However, the corresponding stimulatory effects triggered by HK1 were found to be notably weaker. Furthermore, based on the structural base for HK1, our data suggested that a phenylalanine (F) to valine (V) substitution in the signature motif of HK1 might have contributed to its weak agonistic actions on NKRs and pituitary genes regulation.

Reproductive Regulation of PrRPs in Teleost: The Link Between Feeding and Reproduction.

Prolactin-releasing peptide (PrRP), a sort of vital hypothalamic neuropeptide, has been found to exert an enormous function on the food intake of mammals. However, little is known about the functional role of PrRP in teleost. In the present study, two PrRP isoforms and four PrRP receptors were isolated from grass carp. Ligand-receptor selectivity displayed that PrRP1 preferentially binds with PrRP-R1a and PrRP-R1b, while PrRP-R2a and PrRP-R2b were special receptors for PrRP2. Tissue distribution indicated that both PrRPs and PrRP-Rs were highly expressed in the hypothalamus-pituitary-gonad axis and intestine, suggesting a latent function on food intake and reproduction. Using grass carp as a model, we found that food intake could significantly induce hypothalamus PrRP mRNA expression, which suggested that PrRP should be also an anorexigenic peptide in teleost. Interestingly, intraperitoneal (IP) injection of PrRPs could significantly induce serum luteinizing hormone (LH) secretion and pituitary LHß and GtHα mRNA expression in grass carp. Moreover, using primary culture grass carp pituitary cells as a model, we further found that PrRPs could directly induce pituitary LH secretion and synthesis mediated by AC/PKA, PLC/IP3/PKC, and Ca2+/CaM/CaMK-II pathways. Finally, estrogen treatment of prepubertal fish elicited increases in PrRPs and PrPR receptors expression in primary cultured grass carp hypothalamus cells, which further confirmed that the PrRP/PrRPR system may participate in the neuroendocrine control of fish reproduction. These results, taken together, suggest that PrRPs might act as a coupling factor in feeding metabolism and reproductive activities in teleost.

NK3R Mediates the EGF-Induced SLα Secretion and mRNA Expression in Grass Carp Pituitary.

Epidermal growth factor (EGF) is a potent regulator of cell function in many cell types. In mammals, the EGF/EGFR system played an important role in both pituitary physiology and pathology. However, it is not clear about the pituitary action of EGF in lower vertebrates. In this study, using grass carp as a model, we found that EGF could stimulate NK3R mRNA and protein expression through pituitary ErbB1 and ErbB2 coupled to MEK/ERK and PI3K/Akt/mTOR pathways. In addition, EGF could also induce pituitary somatolactin α (SLα) secretion and mRNA expression in a dose- and time-dependent manner in vivo and in vitro. The stimulatory actions of EGF on SLα mRNA expression were also mediated by PI3K/Akt/mTOR and MEK/ERK pathways coupled to ErbB1 and ErbB2 activation. Our previous study has reported that neurokinin B (NKB) could also induce SLα secretion and mRNA expression in carp pituitary cells. In the present study, interestingly, we found that EGF could significantly enhance NKB-induced SLα mRNA expression. Further studies found that NK3R antagonist SB222200 could block EGF-induced SLα mRNA expression, indicating an NK3R requirement. Furthermore, cAMP/PKA inhibitors and PLC/PKC inhibitors could both abolish EGF- and EGF+NKB-induced SLα mRNA expression, which further supported that EGF-induced SLα mRNA expression is NK3R dependent.

Pituitary Action of E2 in Prepubertal Grass Carp: Receptor Specificity and Signal Transduction for Luteinizing Hormone and Follicle-Stimulating Hormone Regulation.

17ß-estradiol (E2) is an important sex steroid produced by ovary and brain. In mammals, E2 plays an important role in hypothalamus-pituitary-gonad axis to regulate puberty onset, however, little is known about the functional role of E2 in teleost pituitary. Using prepubertal grass carp as model, three nuclear estrogen receptors (nERs: estrogen receptor alpha, estrogen receptor beta 1, and estrogen receptor beta 2) and two G protein-coupled estrogen receptors (GPER1: GPER1a and GPER1b) were isolated from grass carp pituitary. Tissue distribution analysis indicated that both nERs and GPERs were highly detected in grass carp pituitary, which suggested that E2 should play an important role in grass carp pituitary. Using primary cultured grass carp pituitary cells as model, high-throughput RNA-seq was used to examine the E2-induced differentially expressed genes (DEGs). Transcriptomic analysis showed that E2 could significantly upregulate the expression of 28 genes in grass carp pituitary cells, which were characterized into different functions including reproduction, gonad development, and central nervous system development. Further studies confirmed that E2 could induce luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion and mRNA expression in prepubertal grass carp pituitary in vivo and in vitro. In the pituitary, LH and FSH regulation by E2 were mediated by both ERß and GPER1. Apparently, E2-induced LHß and FSHß mRNA expression were mediated by adenylyl cyclase/cAMP/protein kinase A, phospholipase C/inositol 1,4,5-triphosphate/protein kinase C, and Ca2+/calmodulin/CaM-dependent protein kinase II pathways. In addition to LH and FSH, E2 could also induce growth regulation by estrogen in breast cancer 1 (a novel regulator for pituitary development) mRNA expression in grass carp pituitary cells. These results, as a whole, suggested that E2 could play an important role in gonadotropin hormone release and pituitary development in prepubertal grass carp.