Identification, localization and expression of LPXRFamide peptides, and melatonin-dependent induction of their precursor mRNA in the newt brain

Abstract

The existence of RFamide peptides with a C-terminal LPXRFamide (XZL or Q) motif has been identified in the brain of various vertebrate species. However, the presence of LPXRFamide peptides in the urodele brain is not yet known. In this study, we cloned a cDNA encoding the precursor of LPXRFamide peptides from the newt brain by a combination of 3 0 and 5 0 rapid amplification of cDNA ends. The deduced LPXRFamide peptide precursor consisted of 233 amino acid residues, encoding four putative LPXRFamide peptides. All the peptide sequences were flanked by a glycine C-terminal amidation signal and basic amino acid on each end as an endoproteolytic site. Mass spectrometric analyses detected a nonapeptide, two decapeptides and an octapeptide produced from the precursor polypeptide in the brain as endogenous ligands. In situ hybridization further revealed the cellular localization of newt LPXRFamide (nLPXRFa) precursor mRNA in the suprachiasmatic nucleus (SCN) in the newt hypothalamus. Immunocytochemistry showed a cluster of cell bodies restricted to the SCN and their terminals in the median eminence. To understand the regulatory mechanism of nLPXRFa peptide expression, we further analyzed the effect of melatonin on the expression of nLPXRFa precursor mRNA. Melatonin administration to newts increased the expression of nLPXRFa precursor mRNA in the diencephalon. These results indicate that the urodele hypothalamus possesses LPXRFamide peptides and the expression of LPXRFamide peptides is regulated by melatonin. The localization of nLPXRFa peptides further suggests that these peptides may be involved in the regulation of pituitary hormone release in newts.

Molecular, cellular, morphological, physiological and behavioral aspects of gonadotropin-inhibitory hormone

“…fGRP was independently isolated from the European green frog and named Rana RFamide (R-RFa) (Chartrel et al, 2002) (Table 1). In the newt, LPXRFamide peptide cDNA was cloned from the brain of Japanese red-bellied newt (Chowdhury et al, 2011). The deduced precursor also encompasses four LPXRFamide peptides (nLPXRFa-1, -2, -3, -4), and these peptides were also identified as endogenous mature peptides (Chowdhury et al, 2011) (Table 1).…”

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“…All the identified and putative GnIHs in these vertebrates also possess a common C-terminal LPXRFamide (X = L or Q) motif, like avian, mammalian and primate GnIHs (Chartrel et al, 2002;Chowdhury et al, 2011;Koda et al, 2002;Sawada et al, 2002aSawada et al, , 2002bShahjahan et al, 2011;Ukena et al, 2003a). Thus, GnIH peptides exist in the brain of vertebrates from fish to humans (for reviews, see Tsutsui, 2009;Tsutsui and Ubuka, 2012;Tsutsui and Ukena, 2006;Tsutsui et al, 2006bTsutsui et al, , 2007Tsutsui et al, , 2010aTsutsui et al, ,b, 2012bTsutsui et al, , 2013dUkena and Tsutsui, 2005).…”

Neuroanatomical Organization of the Brain Gonadotropin-Inhibitory Hormone and Gonadotropin-Releasing Hormone Systems in the Frog <b><i>Pelophylax esculentus</i></b>

“…Indeed, the precursor polypeptide encompasses fGRP and its gene-related peptides (fGRP-RP-1, fGRP-RP-2, and fGRP-RP-3), which were all identified as mature peptides . GnIH orthologs (LPXRFamide peptides) were also assessed in the brain of the Japanese red-bellied newt Cynops pyrrhogaster, a urodele species [Chowdhury et al, 2011].…”

“…The general pattern of the localization of these peptides seems to be evolutionarily conserved in some brain areas, as demonstrated by both immunohistochemical and in situ hybridization studies. Specifically, the presence of neurons containing GnIH and its orthologs (LPXRFamide peptides) in the periventricular region of the hypothalamus appears to be a common feature in mammals [Hinuma et al, 2000;Fukusumi et al, 2001;Yano et al, 2003;Kriegsfeld et al, 2006;Johnson et al, 2007;Clarke et al, 2008;Legagneux et al, 2009;Ubuka et al, 2009aUbuka et al, , b, 2012, birds [Tsutsui et al, 2000;Bentley et al, 2003;Ubuka et al, 2003Ubuka et al, , 2008Osugi et al, 2004;Tobari et al, 2010], amphibians [Chartrel et al, 2002;Koda et al, 2002;Sawada et al, 2002a;Ukena et al, 2003;Chowdhury et al, 2011], and fish [Sawada et al, 2002b;Amano et al, 2006]. In contrast to the highly localized clusters of cell bodies, GnIH-positive nerve fibers are widely distributed across brain areas in all of the vertebrate species that have been studied [for reviews, see Tsutsui, 2009;Tsutsui et al, 2010aTsutsui et al, , b, 2012.…”