ABSTRACT
Secretoneurin (SN) is an important stimulator of pituitary luteinizing hormone (LH) synthesis and secretion in goldfish. It is unknown whether this neuropeptide performs the same role in other fish species. In this study, the full-length cDNAs encoding Secretogranin IIa (SgIIa) and b (SgIIb) were cloned from the brain of orange-spotted grouper. Sequence analysis showed that a 34-amino acid SN peptide (SNa) is present in SgIIa proprotein, and a 33-amino acid SN peptide (SNb) is present in SgIIb proprotein. The two SN peptides share a low degree of similarity but contain the signature YTPQ-X-LA-X7-EL sequence. Real-time PCR showed that two SgII genes are mainly expressed in the brain and pituitary. During ovarian development, the expression levels of two SgII genes in the hypothalamus and pituitary were significantly reduced at the stage when the ovary contained full-grown oocytes. The biological functions of the two SN peptides were further investigated in vitro and in vivo. Both SN peptides could significantly elevate the mRNA levels of Gonadotropin-Releasing Hormone 1 (GnRH1) and 3 (GnRH3) in the hypothalamic fragments and upregulated the expression of Follicle-Stimulating Hormone beta (FSHb) and Luteinizing Hormone beta (LHb) in the pituitary cells. The stimulatory effects on the expression of GnRHs and Gonadotropins were also observed after intraperitoneal injection of SN peptides. Our study indicated that the SgII/SN system has stimulatory effects on the reproductive axis of orange-spotted grouper.
Subject(s)
Bass/genetics , Reproduction/genetics , Secretogranin II/genetics , Secretogranin II/physiology , Amino Acid Sequence , Animals , Bass/metabolism , Cloning, Molecular , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Female , Gene Expression Profiling , Male , Secretogranin II/isolation & purification , Secretogranin II/metabolism , Sequence Analysis, DNAABSTRACT
Secretoneurin (SN) is a 33- to 34-amino acid neuropeptide derived from secretogranin-II, a member of the chromogranin family. We previously synthesized a putative goldfish (gf) SN and demonstrated its ability to stimulate LH release in vivo. However, it was not known whether goldfish actually produced the free SN peptide or whether SN directly stimulates LH release from isolated pituitary cells. Using a combination of reverse-phase HPLC and mass spectrometry analysis, we isolated for the first time a 34-amino acid free gfSN peptide from the whole brain. Moreover, Western blot analysis indicated the existence of this peptide in goldfish pituitary. Immunocytochemical localization studies revealed the presence of SN immunoreactivity in prolactin cells of rostral pars distalis of the anterior pituitary. Additionally, we found that magnocellular cells of the goldfish preoptic region are highly immunoreactive for SN. These neurons send heavily labeled projections that pass through the pituitary stalk and innervate the neurointermediate and anterior lobes. In static 12-h incubation of dispersed pituitary cells, application of SN antiserum reduced LH levels, whereas 1 and 10 nM gfSN, respectively, induced 2.5-fold (P < 0.001) and 1.9-fold (P < 0.01) increments of LH release into the medium, increases similar to those elicited by 100 nM concentrations of GnRH. Like GnRH, gfSN elevated intracellular Ca(2+) in identified gonadotrophs. Whereas we do not yet know the relative contribution of neural SN or pituitary SN to LH release, we propose that SN could act as a neuroendocrine and/or paracrine factor to regulate LH release from the anterior pituitary.
