RFamide peptides inhibit the expression of melanotropin and growth hormone genes in the pituitary of an Agnathan, the sea lamprey, Petromyzon marinus.

Neuropeptides with the Arg-Phe-amide motif at their C termini (RFamide peptides) were identified in the brains of several vertebrates, and shown to have important physiological roles in neuroendocrine, behavioral, sensory, and autonomic functions. The present study identified RFamide peptides, which are teleost prolactin-releasing peptide (PrRP) homologs, in the sea lamprey, Petromyzon marinus and characterized their effect on the release of pituitary hormones in vitro. Two RFamide peptides (RFa-A and RFa-B) were isolated from an acid extract of sea lamprey brain, including hypothalamus by Sep-Pak C18 cartridge, affinity chromatography using anti-salmon PrRP serum, and reverse-phase HPLC on an ODS-120T column. Amino acid (aa) sequences and mass spectrometric analyses revealed that RFa-A and RFa-B consist of 25 and 20 aa, respectively, and have 75% sequence identity within the C-terminal 20 aa. The RFa-B cDNA encoding a preprohormone of 142 aa was cloned from the lamprey brain, and the deduced aa sequence from positions 48-67 was identical to the sequence of RFa-B. However, the preprohormone does not include an aa sequence similar to the RFa-A sequence. Cell bodies, which were immunoreactive to anti-salmon PrRP serum, were located in the periventricular arcuate nucleus, ventral part of the hypothalamus, and immunoreactive fibers were abundant from the hypothalamus to the brain. A small number of immunoreactive fibers were detected in the dorsal half of the rostral pars distalis of the pituitary, close to the GH-producing cells. In addition, anti-salmon PrRP immunoreactivities were observed in the pars intermedia, corresponding to melanotropin cells. Likewise, signal of RFa-B mRNA was detected not only in the brain but also in the pars intermedia. The synthetic RFa-A and -B inhibited GH mRNA expression in a dose-dependent fashion in vitro, which is comparable to the inhibitory effect of teleost PrRP on GH release. Both RFa-A and -B also inhibited the expression of proopiomelanotropin mRNA, but no effects were observed in the expression of proopiocortin and gonadotropin beta mRNAs. The results indicate that RFamide peptides, which are teleost PrRP homologs, are present in the hypothalamus and pituitary of sea lamprey, and may be physiologically involved in the inhibition of GH and melanotropin release in the sea lamprey pituitary.

Identification of sea lamprey GTHbeta-like cDNA and its evolutionary implications.

We have identified the first and perhaps only gonadotropin beta-like protein by cDNA cloning in sea lamprey, a member of the oldest lineage of vertebrates, the agnathans. Two pituitary gonadotropins (GTHs: follicle-stimulating hormone (FSH) and luteinizing hormone (LH)) have been identified in representative species of all classes of vertebrates except the agnathans. The present study was undertaken to identify GTH in sea lamprey, Petromyzon marinus, to gain a further understanding of the origin and evolution of reproductive pituitary hormones and their respective genes in vertebrates. Sea lamprey preGTHbeta-like cDNA was cloned from a plasmid cDNA library using an expressed sequence tag analysis. The preGTHbeta-like cDNA encoded 150 amino acids, in which the GTHbeta-like protein consisted of 134 amino acid residues. Sea lamprey GTHbeta-like protein contained 12 Cys residues and two N-glycosylation sites at homologous positions to those of FSHbeta and LHbeta. The region of the molecule that has been proposed to control receptor binding specificity (i.e., the region between the 10th and 12th Cys residues) suggests that the proposed heterodimer would be more like a FSH than a LH. Sea lamprey GTHbeta-like protein-producing cells were identified immunocytochemically in the ventral part of the proximal pars distalis of pituitary using antiserum prepared against a synthetic peptide of preGTHbeta-like protein (52-68). Intraperitoneal administration of sea lamprey GnRH-I and -III at 100 microg/g body weight (twice at a 24h interval) increased expression of GTHbeta-like protein in the pituitary of adult female sea lamprey during the final maturational period. Thus, these results are the first to demonstrate the presence of a single GTH-like system in lampreys. Because the sea lamprey GTHbeta-like protein is a clear out-group compared to those of the LH and FSH family based on phylogenic analysis, we propose that an ancestral glycoprotein hormone gave rise to only one GTH in lampreys and to the glycoprotein hormone family that gave rise to LH, FSH, and TSH during the early evolution of gnathostomes.

Structures for the proopiomelanocortin family genes proopiocortin and proopiomelanotropin in the sea lamprey Petromyzon marinus.

Gnathostomes express a common proopiomelanocortin (POMC) gene in the pars distalis (PD) and the pars intermedia (PI) of the pituitary gland. In contrast, the sea lamprey Petromyzon marinus expresses one distinct gene in each lobe; proopiocortin (POC) encoding adrenocorticotropic hormone (ACTH) and beta-endorphin (END) is expressed in the PD and proopiomelanotropin (POM) encoding melanophore-stimulating hormone (MSH), and a different beta-END is expressed in the PI. We characterized the genomic structure of the sea lamprey POC and POM genes including their 5'-flanking regions. Both genes have two introns at positions similar to those of gnathostomes. Each exon encodes genetic information seen in the gnathostome POMC gene: exon 1 encodes an untranslated nucleotide sequence, exon 2 encodes a signal peptide and the N-terminal short part of POC or POM, and exon 3 encodes all other parts including ACTH, MSHs or beta-END. Intron-A of POM (2289 bp) is six times longer than that of POC (379 bp). The POM intron-A has three transposon-like sequences (TnL-1, -2, -3), the total length of which is 1781 bp, suggesting that it has expanded via the insertion of TnLs. The 5'-flanking region of the POC gene contains two TATA boxes, a CCAAT box, eight E boxes, STAT, RAIE, and one binding site each for Ptx1, Pit-1, and Tpit. The POM gene contains four TATA boxes, eight E boxes, three STATs, two RAIEs, two CRE-like elements, and one binding site for Pit1. However, there is virtually no similarity between the two genes in the distribution of the elements. The transcriptional regulation of POC and POM may have diverged with the functional differentiation of the two genes.