Secretoneurin (SN) is a 31-43 amino acid, functional peptide derived by proteolytic processing from the middle domain of the ~600 amino acid secretogranin-II (SgII) precursor. In teleosts there are 2 forms arising from 2 different genes, SgIIa and SgIIb. In turn, there are both SNa and SNb in teleost. Secretoneurin is a well-conserved peptide during evolution from fish to mammals and widely distributed in secretory granules of endocrine cells and neurons. Secretoneurin plays important roles in different biological processes, for example controlling vertebrate reproduction by stimulating luteinizing hormone release from the pituitary. A potential new role of SN in feeding in goldfish is the subject of the research presented in this thesis. Firstly, we looked at the distribution of SgIIa mRNA in various female goldfish tissues using both RT-PCR and Q-PCR techniques in order to determine which tissue expresses SgIIa mRNA and in which level. We found that SgIIa mRNA was detected in different amounts in all tissues examined. The main tissues of interest were hypothalamus, telencephalon and gut, they all expressed SgIIa. Secondly, we examined the effect of acute (26 h), short (3 days), medium (7 days) and long (14 days) fasting and periprandial changes on SgIIa mRNA level in hypothalamus, telencephalon and gut using Q-PCR method. The results showed that SgIIa mRNA increases under the effect of acute and short fasting, however, medium and long fasting did not affect SgIIa mRNA. Thirdly, we examined the effect of brain injection of goldfish SNa on food intake and locomotor behavior and the expression of some feeding neuropeptides such as neuropeptide Y, orexin, chocystokinin and cocaine-and amphetamine-regulated transcript I after treatment. Injection of SNa in the third brain ventricle increased food intake and fish activity. Associated with this was an increase in NPY and decrease in CARTI mRNA levels in hypothalamus. The increase in SgIIa mRNA following fasting and the increase of food intake as a result of SNa treatment suggest a novel role for SNa in feeding processes.

Secretoneurin (SN) is a 31-43 amino acid, functional peptide derived by proteolytic processing from the middle domain of the ~600 amino acid secretogranin-II (SgII) precursor. In teleosts there are 2 forms arising from 2 different genes, SgIIa and SgIIb. In turn, there are both SNa and SNb in teleost. Secretoneurin is a well-conserved peptide during evolution from fish to mammals and widely distributed in secretory granules of endocrine cells and neurons. Secretoneurin plays important roles in different biological processes, for example controlling vertebrate reproduction by stimulating luteinizing hormone release from the pituitary. A potential new role of SN in feeding in goldfish is the subject of the research presented in this thesis. Firstly, we looked at the distribution of SgIIa mRNA in various female goldfish tissues using both RT-PCR and Q-PCR techniques in order to determine which tissue expresses SgIIa mRNA and in which level. We found that SgIIa mRNA was detected in different amounts in all tissues examined. The main tissues of interest were hypothalamus, telencephalon and gut, they all expressed SgIIa. Secondly, we examined the effect of acute (26 h), short (3 days), medium (7 days) and long (14 days) fasting and periprandial changes on SgIIa mRNA level in hypothalamus, telencephalon and gut using Q-PCR method. The results showed that SgIIa mRNA increases under the effect of acute and short fasting, however, medium and long fasting did not affect SgIIa mRNA. Thirdly, we examined the effect of brain injection of goldfish SNa on food intake and locomotor behavior and the expression of some feeding neuropeptides such as neuropeptide Y, orexin, chocystokinin and cocaine-and amphetamine-regulated transcript I after treatment. Injection of SNa in the third brain ventricle increased food intake and fish activity. Associated with this was an increase in NPY and decrease in CARTI mRNA levels in hypothalamus. The increase in SgIIa mRNA following fasting and the increase of food intake as a result of SNa treatment suggest a novel role for SNa in feeding processes.

At the hypothalamo-pituitary interface, multiple neurotransmitters and neuropeptides interact to control luteinizing hormone and follicle stimulating hormone release from gonadotrophs. The luteinizing hormone surge is essential for fertility as it triggers ovulation in females and sperm release in males. While it is well-established that gonadotropin-releasing hormone and/or kisspeptin are required for pulsatile and surge release of luteinizing hormone in mammalian species, their essentiality is challenged by studies showing knockouts in zebrafish and medaka do not block reproduction. In goldfish, secretoneurin-a, a neuropeptide derived from secretogranin-IIa processing, stimulates luteinizing hormone release in vivo and from dispersed pituitary cells in vitro. Secretoneurin does not bind to the human gonadotropin releasing hormone receptor and can enhance luteinizing hormone release when applied directly to mouse LbetaT2 cells. Our study indicates the presence of secretogranin-IIa and secretogranin-IIb mRNA in specific regions of the zebrafish brain and pituitary that have been implicated in the control of reproductive processes. I also observed that secretogranin-II knockout disrupts normal morphology of the pectoral fins by reducing the number of breeding tubercule rows, breeding tubercule width and breeding tubercule cluster length which could potentially lead to a reduced spawning success. Knocking out the secretogranin-IIa and secretogranin-IIb genes using TALENs in zebrafish indicates that these genes are required for optimal reproduction. Rates of oviposition for double secretogranin-II knockout females are 6% and 11% when crossed with double secretogranin-II knockout and wild-type males, respectively, compared to 62% in virgin wild-type pairings. Comprehensive video analysis demonstrates that secretogranin-II knockout reduces all stereotypical male courtship behaviours. Severe reductions in the expression of gonadotropin releasing hormone 3 in the hypothalamus and luteinizing hormone in the pituitary suggest that secretogranin-II-derived peptides drive the gonadotropin releasing hormone-luteinizing hormone control system. Spawning success is rescued in double secretogranin-II knockouts following one injection of synthetic secretoneurin-a in which it increases from 11% to 30% thereby supporting the hypothesis that secretoneurin-a is a key reproductive hormone. However, embryo survival rate of secretoneurin-a injected double secretogranin-II knockout was lower than saline-injected wild-type pairings. Injection of human chorionic gonadotropin, a hormone that mimics the action of luteinizing hormone on binding and activating the luteinizing hormone receptor in fish, increased double secretogranin-II knockout spawning success to 38%, thus comparing favourably to 47% in saline-injected wild-type controls. My data provides support that secretogranin-II is required for optimal reproductive functions although the precise mechanisms behind this reduced capacity in zebrafish lacking the secretogranin-II genes remain to be investigated. Moreover, the high conservation of secretoneurin from lamprey to human suggests a broader importance of this emerging peptide family.

Secretoneurin (SN) is a 31-42 amino acid neuropeptide, derived from the proteolytic processing of the precursor protein secretogranin-II (Scg2). In zebrafish, SNa and SNb are respectively 34 and 31 amino acids long, deriving from selective processing of the distinct Scg2a and Scg2b precursors. Our lab recently reported that frameshift mutations in Scg2 leads to reduces sexual behavior and disrupted spawning. This defect was partially rescued by injection of SNa. In my work, we determined the distribution of SNa in relation to other known reproductive hormones in zebrafish brain and pituitary by double immunofluorescent staining. SNa-immunoreactivity (ir) was observed in neuronal cell bodies in the ventral telencephalon, preoptic area (POA) and hypothalamus. Neuronal fibers staining for SNa projecting from the magnocellular POA passed through the pituitary stalk and terminated largely in the neurointermediate lobe (NIL). The SNa-ir fibers were less abundant but clearly present in the pars distalis. Moreover, SNa colocalized with isotocin in cell bodies in the POA and fibers in the NIL. Using the lhb-RFP x fshb-eGFP transgenic zebrafish line, we observed SNa-ir near gonadotroph cell bodies but not in them. Peptidomic analysis uncovered shorter processed fragments of both in SNa and SNb in whole brain and pituitary. We performed mass spectrometry to determine natural periovulatory variations and studied their potential bioactivities. Both SNa1-34 and SNa1-14 in the brain varied during the ovulatory cycle, while SNb-related peptides were relatively stable. The levels of SNa1-34 in brain peaked coincident with increased Gnrh3 at the time of the luteinizing hormone (Lh) surge. The levels of SNa1-14 in brain and ovaries peaked at the time of ovulation. To investigate the potential bioactivity of SNa1-34 and SNa1-14, we performed intraperitoneal injections and analyzed the expression numerous reproductive genes. The results suggested that SNa1-34 could induce ovulation by stimulating time-dependent expression of gnrh3 in brain, cga and lhb in pituitary and npr in ovaries. In contrast, SNa1-14 exhibited far fewer effects, but stimulated the expression of gnrh2 but suppressed gnrhr2, so its natural biological function remains unknown. After a single injection of SNa1-34 in females isolated from males, 61% (11/18) zebrafish ovulated. This compares favorably with the effects of the Lh analog human chorionic gonadotropin, inducing ovulation in 72% (13/18) of females. Natural variations in levels of SN in relation to other well-known neuropeptides and biological activity data in the zebrafish model support the hypothesis that SNa is a new stimulatory reproductive hormone. The SN peptides are conserved in evolution so what we uncover in fish may help us speculate on its importance in other vertebrates.