Characterization and Role of Secretoneurin in the Ovulatory Cycle of Zebrafish
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.
Secretogranin 2, Secretoneurin, Zebrafish, Ovulation, Brain, Pituitary
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Université d'Ottawa / University of Ottawa