Gonadotropin (GTH)-inhibitory hormone (GnIH) is a novel hypothalamic neuropeptide that inhibits GTH secretion in mammals and birds by acting on gonadotropes and GnRH neurons within the hypothalamic-pituitary-gonadal axis. GnIH and its orthologs that have an LPXRFamide (X = L or Q) motif at the C terminus (LPXRFamide peptides) have been identified in representative species of gnathostomes. However, the identity of an LPXRFamide peptide had yet to be identified in agnathans, the most ancient lineage of vertebrates, leaving open the question of the evolutionary origin of GnIH and its ancestral function(s). In this study, we identified an LPXRFamide peptide gene encoding three peptides (LPXRFa-1a, LPXRFa-1b, and LPXRFa-2) from the brain of sea lamprey by synteny analysis and cDNA cloning, and the mature peptides by immunoaffinity purification and mass spectrometry. The expression of lamprey LPXRFamide peptide precursor mRNA was localized in the brain and gonad by RT-PCR and in the hypothalamus by in situ hybridization. Immunohistochemistry showed appositions of lamprey LPXRFamide peptide immunoreactive fibers in close proximity to GnRH-III neurons, suggesting that lamprey LPXRFamide peptides act on GnRH-III neurons. In addition, lamprey LPXRFa-2 stimulated the expression of lamprey GnRH-III protein in the hypothalamus and GTHβ mRNA expression in the pituitary. Synteny and phylogenetic analyses suggest that the LPXRFamide peptide gene diverged from a common ancestral gene likely through gene duplication in the basal vertebrates. These results suggest that one ancestral function of LPXRFamide peptides may be stimulatory compared with the inhibitory function seen in later-evolved vertebrates (birds and mammals).
GnRH had long been considered the only major hypothalamic regulator of pituitary gonadotropin (GTH) secretion in vertebrates. The recent discovery of GTH-inhibitory hormone (GnIH) that inhibits GTH secretion by acting on gonadotropes and GnRH neurons in mammals and birds has now changed our classical understanding of the regulation of hypothalamic-pituitary-gonadal (HPG) axis in vertebrates (for reviews, see Refs. 1–3). GnIH was originally identified in the quail brain (1). GnIH was shown to be located in the hypothalamo-hypophysial system (1–7) and to decrease GTH secretion from the pituitary (1–3, 6, 8) in several avian species. After the discovery of GnIH, its orthologous peptides have been identified in a variety of vertebrates, such as mammals [RFamide-related peptides (RFRP)] (9–12), amphibians [frog growth hormone-releasing peptide (fGRP) (13, 14), Rana RFamide (15), and newt LPXRFa peptide (16)] and teleosts [goldfish (gf)LPXRFa (17)]. All the identified peptides possessed a C-terminal Leu-Pro-Xaa-Arg-Phe-NH2 (Xaa = Leu or Gln) motif at their C termini, and thus, they were designated as LPXRFamide (X = L or Q) peptides (LPXRFa peptides). Accordingly, LPXRFa peptides constitute one of the largest groups in the RFamide peptide (RFa peptide) family in vertebrates (for reviews, see Refs. 2, 3, 18). Typically, the LPXRFa peptide precursors in bony vertebrates encode two to four LPXRFa peptides, such as RFRP-1 and RFRP-3 in mammals (10–12), GnIH, GnIH gene-RP-1 and GnIH-RP-2 in birds (4, 6, 7, 19), fGRP, fGRP-RP-1, fGRP-RP-2, and fGRP-RP-3 in amphibians (20), and LPXRFa-1, LPXRFa-2, and LPXRFa-3 in fish (17, 21, 22). Studies on teleost fish and amphibians have shown that functions of LPXRFa peptides in earlier evolved vertebrates were stimulatory or inhibitory (13, 14, 21–23). Although the existence of GnIH orthologs (LPXRFa peptides) was demonstrated in gnathostomes (jawed vertebrates) from teleosts to humans, GnIH orthologs (LPXRFa peptides) had yet to be identified in lampreys, one of the only two extant members of the oldest lineage of vertebrates extending back over 500 million years (24). Therefore, the objective of our study was to identify GnIH orthologs (LPXRFa peptides) in lampreys to determine their expression and function and to analyze the evolution of this family through phylogenetic and synteny analysis.
From a structural point of view, PQRFa peptides, pain modulatory neuropeptides that include neuropeptide FF and neuropeptide AF, share a C-terminal Pro-Gln-Arg-Phe-NH2 motif (for a review, see Ref. 25). Interestingly, the C-terminal motifs of LPXRFa and PQRFa peptides, which are considered to be important for the interaction with their receptors [G-protein coupled receptor (GPR) GPR147 and GPR74], as well as the structure of their receptors showed high sequence similarities (for reviews, see Refs. 2, 3, 18, 25), suggesting that an LPXRFa peptide gene and a PQRFa peptide gene may have diverged from a common ancestral gene through gene duplication.
Lampreys and hagfish are the only two extant members of the superclass Agnatha, the most basal vertebrates. Lampreys are the earliest evolved vertebrates, for which there is a demonstrated neuroendocrine system. Sexual maturation and reproduction are seasonal and synchronized processes in the sea lamprey controlled by the hypothalamus-pituitary axis (26–28). Three hypothalamic forms of GnRH, lamprey GnRH-I, GnRH-II, and GnRH-III, have been identified in the sea lamprey and shown by extensive physiological, anatomical, and immunological studies to control reproduction (28–30). In contrast to gnathostomes, in which two GTH have been identified, only one pituitary glycoprotein hormone, GTHβ, has been identified in the sea lamprey, and its mRNA expression was shown to be stimulated by GnRH-I or GnRH-III (31). The molecular, biochemical, and functional studies of GnRH, GTH, and respective receptors show that these neuroendocrine factors share common functional and developmental features compared with later evolved vertebrates (32, 33). We previously identified PQRFa peptides from the brains of sea lamprey (lamprey PQRFa) (34) and brown hagfish (hagfish PQRFa) (35). Here, we show that sea lampreys possess the LPXRFa peptide gene, and its mature functional peptide acts on the HPG axis. This study provides novel insights into the evolutionary history of GnIH and its function through vertebrate evolution.
Materials and Methods
Animals
Adult sea lampreys were collected from fish ladders in the Cocheco River in Dover and Exeter, NH, and maintained at the Anadromous Fish and Aquatic Invertebrate Research laboratory at University of New Hampshire in May and June of 2009 and 2010 during their spawning migration from the sea to freshwater (as described in Ref. 27). Lampreys were held in flow-through freshwater tanks. Water temperature in the tanks was maintained at ambient temperatures ranging between 9 and 18 C during the spawning season. The experimental protocols were approved by the committee of Waseda University, the University of New Hampshire, and Niigata University and performed according to the University of New Hampshire Institutional Animal Care and Use guidelines and to the Guide for the Care and Use of Animals prepared by the Waseda University and Niigata University.
Genome database search
It was reported that a conserved synteny region exists around the LPXRFa peptide gene loci in the chromosome of some vertebrates (21, 36). In that region, the cytochrome c (CYCS) gene is located near the LPXRFa peptide gene (21, 36). Therefore, we searched for a putative lamprey CYCS gene using TBLASTN program in Ensembl Genome Browser (http://www.ensembl.org/Petromyzon_marinus/Info/Index/). We then analyzed the nucleotide sequences around the putative lamprey CYCS gene using GENSCAN (http://genes.mit.edu/GENSCAN.html) (37) to search for a putative lamprey LPXRFa peptide precursor gene. Subsequently, we performed the genome synteny analysis by comparing the gene loci of LPXRFa peptide precursors in lamprey, human, chicken, Xenopus tropicalis, and zebrafish using Ensembl Genome Browser (http://www.ensembl.org/index.html). We also performed the genome synteny analysis by comparing the gene loci of PQRFa peptide precursors in human, anole lizard, X. tropicalis, and zebrafish.
Molecular cloning
Total RNA was extracted from the lamprey brains using ISOGEN (NIPPON GENE, Tokyo, Japan) in accordance with the manufacturer's instructions. First-strand cDNA was synthesized with the oligo(dT)-anchor primer supplied in the 5′/3′ rapid amplification of cDNA ends kit (Roche Diagnostics, Basel, Switzerland). We found a putative lamprey LPXRFa peptide precursor gene in the scaffold GL476598 (to be updated to scaffold 270) in the lamprey genome database (http://www.ensembl.org/Petromyzon_marinus/Info/Index). Therefore, we performed a cDNA cloning based on the nucleotide sequences of the scaffold GL476598 (to be updated to scaffold 270). The sequence of lamprey LPXRFa peptide precursor cDNA was determined as described previously (34, 35). The detail method of cDNA cloning is described in Supplemental Materials and Methods (published on The Endocrine Society's Journals Online web site at http://endo.endojournals.org).
Phylogenetic analysis
Multiple sequence alignments and phylogenetic analyses of the precursor cDNA of lamprey LPXRFa orthologous peptides were performed by using CLUSTAL W 1.83 (European Molecular Biology Laboratory). The phylogenetic tree was constructed by the neighbor-joining method. The data were obtained from 1000 bootstrap replicates to determine the confidence indices within the phylogenetic tree. The bootstrap values were shown as percentage after 1000 replications on the branches. The GenBank accession nos. of the sequences used in the phylogenetic analysis are shown in Supplemental Table 1.
Peptide extraction and immunoaffinity purification
The brains of approximately 500 adult sea lampreys were collected, immediately frozen on dry ice, and stored at −80 C until used. The brains were boiled and homogenized in 5% acetic acid as described previously (34, 35). The homogenate was centrifuged at 10,000 × g for 30 min at 4 C, and the resulting precipitate was again homogenized and centrifuged. The two supernatants were pooled and concentrated by using a rotary evaporator at 40 C. After precipitation with 75% acetone, the supernatant was passed through a disposable C18 cartridge column (Mega Bond-Elut; Varian, Harbor City, CA), and the retained material eluted with 60% methanol was loaded onto an immunoaffinity column. The affinity chromatography was performed as described previously (34, 35). The antiserum against PQRFa peptide (lamprey PQRFa) (34) was conjugated to Protein A Sepharose 4B (Amersham Pharmacia Biotech, Uppsala, Sweden) as an affinity ligand. The brain extract was applied to the column at 4 C, and the adsorbed materials were eluted with 0.3 m acetic acid containing 0.1% 2-mercaptoethanol. An aliquot of each fraction (1 ml) was analyzed by a dot immunoblot assay with the antiserum against PQRFa peptide according to our previous methods (34, 35).
HPLC and structure determination
The immunoreactive materials obtained by immunoaffinity purification using the antiserum against PQRFa peptide were subjected to a HPLC column (ODS-80TM; Tosoh, Tokyo, Japan) with a linear gradient of 10–50% acetonitrile containing 0.1% trifluoroacetic acid for 100 min at a flow rate of 0.5 ml/min, and the eluted fractions were collected every 2 min and assayed by immunoblotting. The fractions corresponding to the elution times of 44–46 min (see figure 4 below) and 46–48 min (see figure 4 below) showed intense immunoreactivities. Each immunoreactive fraction was then loaded onto another reverse-phase column (ODS-80TM; Tosoh) under an isocratic condition of 22% acetonitrile containing 0.1% trifluoroacetic acid for 30 min at a flow rate of 0.5 ml/min (see figure 4 below). The isolated immunoreactive substances were subjected to amino acid sequence analysis by automated Edman degradation with a gas-phase sequencer (PPSQ-10; Shimadzu, Kyoto, Japan). Molecular weight was determined by MALDI-TOF MS (matrix assisted-laser desorption/ionization time-of-flight mass spectrometry) (AXIMA-CFR plus; Shimadzu). The theoretical mass values were calculated according to the amino acid sequence of the synthetic peptides by Protein Prospector version 5.9.0 (http://prospector.ucsf.edu/prospector/mshome.htm).
Tissue distribution of LPXRFa transcript in the lamprey by RT-PCR
Tissue total RNA (brain, pituitary, and gonad) were extracted from three male and three female lampreys with 1.0 ml of QIAzol Lysis reagent (QIAGEN, Venlo, Netherlands) and digested with 5 U of RQ1 ribonuclease-free deoxyribonuclease (DNase) (Promega, Madison, WI) for 1 h at 37 C to remove genomic DNA. RNeasy lipid tissue mini kit (QIAGEN) was used for total RNA extraction of ovaries from three females according to the manufacturer's direction. After the DNase treatment, total RNA was isolated with 0.3 ml of QIAzol Lysis reagent. For RT-PCR analysis, total RNA of each tissue from three males or three females was mixed, respectively. First strand cDNA was reverse transcribed from 3 μg of the total RNA with 100 U of Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA) and 200 ng of NotI-(dt)18 primer according to the manufacturer's protocol. PCR was performed at 95 C for 3 min as an initial denaturation followed by 40 cycles at 95 C for 15 sec and 60 C for 1 min to detect LPXRFa transcript. PCR mixture was composed of 1× Advantage 2 PCR buffer, 1× Advantage 2 Polymerase Mix (CLONTECH, Palo Alto, CA), 0.2 mm deoxynucleotide triphosphate mix, one-tenth of first strand cDNA, 10% dimethylsulfoxide (Sigma, St. Louis, MO) and 500 nm forward primer (la-LPXRFa F1, 5′-ATGTTGGCTGGTTTCCTGCT-3′) and reverse primer (la-LPXRFa R1, 5′-GGATCCCCACCCCTCTTG-3′). PCR amplification of elongation factor (EF)1α was also performed at 35 cycles of the same thermal profile with forward primer [Petromyzon marinus (PM) EF1A open reading frame-F1, 5′-CCTCCATCCATCATGGGCAAGGAAAAG-3′] and reverse primer (PM EF1A R, 5′-ACCGGCCTCAAACTCACCTA-3′) in the same composition of the PCR mixture without dimethylsulfoxide as a positive control.
In situ hybridization
The expression of lamprey LPXRFa peptide precursor mRNA in the brain was localized by in situ hybridization. The dissected brain and attached pituitary were immersed in refrigerated 4% paraformaldehyde in 0.1 m phosphate buffer for about 24 h. Subsequently, the brain with the pituitary were soaked in a refrigerated sucrose solution (30% sucrose in PBS) until they sank. The brains were sectioned frontally at a 10-μm thickness with a cryostat at −20 C. The sections were placed onto 3-aminopropyltriethoxysilane-coated slides. In situ hybridization was carried out according to our previous method (34, 35) using a digoxigenin-labeled antisense RNA probe (for details, see Supplemental Materials and Methods). In the present study, we followed the nomenclature of Pombal et al. (38).
Immunohistochemistry
Antisera were raised against the N terminus of lamprey LPXRFa-2, because the C-terminal PQRFa motif of lamprey LPXRFa peptides is identical to previously identified lamprey PQRFa peptides (34). The antisera were raised according to our previous method (34) using the synthetic N-terminal lamprey LPXRFa-2 sequence with an addition of C-terminal cysteine residue (SEPFWHRTRPQRFC). We also used antisera raised against lamprey GnRH-III as described previously (39). Tissue preparations have been described elsewhere (39). The brains were embedded in Paraplast, and serial frontal or sagittal sections of 6–10 μm were mounted on gelatin-coated glass slides. Immunoreactive products were detected with an ABC kit or ABC-AP kit (Vector Laboratories, Inc., Burlingame, CA), according to our previous method (39). Control for specificity of the immunohistochemistry of antilamprey LPXRFa was performed by preabsorbing the working dilution (1:750) with synthetic lamprey LPXRFa-2 (6 μg/ml) or synthetic lamprey PQRFa (30 μg/ml). The detail method of immunohistochemistry is described in Supplemental Materials and Methods.
In vivo analyses of biological activities
A total of 35 female sea lampreys in each of 2009 and in 2010 were individually injected ip with one of seven different treatments, five lamprey per treatment group. The water temperature during this experiment was 15 C. Each lamprey was injected two times 24 h apart with saline (control), lamprey LPXRFa-1a at 50 or 100 μg/kg, lamprey LPXRFa-1b at 50 or 100 μg/kg, or lamprey LPXRFa-2 at 50 or 100 μg/kg. After 72 h from the first injection, each lamprey was decapitated, and the brain and pituitary were removed and immediately frozen in liquid nitrogen. The samples were stored at −80 C.
Protein extraction, HPLC, and RIA
Each lamprey brain was extracted for GnRH-I, GnRH-II, and GnRH-III as previously described (40) and modified (41, 42). Individual brain extracts were injected into a loop on a HPLC system. This system consists of a PerkinElmer (Waltham, MA) series 100 pump with a Pecosphere 3 CR C18 reverse-phase column (41). The isocratic mobile phase consisted of 7.40 g of ammonium acetate and 3.04 g of citric acid in 1 liter of 19% acetonitrile/water (final pH was adjusted to 4.6 with phosphoric acid); 110 fractions, 1 ml each, were collected for each sample injected (42). The fractions were dried using a Speed Vac system and stored at −20 C.
Radioimmunoassay
Duplicate 100-μl aliquots of the peak fractions were assayed to determine the concentration of lamprey GnRH-I, GnRH-II, and GnRH-III using methods previously described (42, 43). I125 lamprey GnRH (lamprey GnRH-I and GnRH-III assay) and I125 chicken GnRH (lamprey GnRH-II assay) were iodinated, respectively, and appropriate synthetic lamprey GnRH was used as a standard for each of the assays. Antiserum 3952 (lamprey GnRH-III) and 135–66 (lamprey GnRH-II) were used at an initial dilution of 1:16,000 and 1:40,000, respectively. RIA for lamprey GnRH-I and GnRH-III was performed on HPLC fractions 1–15, and RIA for lamprey GnRH-II was performed on HPLC fraction 35–55. Data for hormone concentrations were analyzed for each year using ANOVA, and significant differences between treatment samples and saline samples were determined using Fisher's protected least significant difference (PLSD). After the individual analysis, the data were combined for both years and reanalyzed using ANOVA and Fisher's PLSD. In all tests, the level of significance for different groups was P < 0.05. The detail method of the RIA is described in Supplemental Materials and Methods.
Pituitary GTH expression studies by quantitative PCR
Total RNA from individual pituitary was extracted with QIAzol Lysis reagent (QIAGEN) and digested with RQ1 ribonuclease-free DNase (Promega) for 1 h at 37 C to remove genomic DNA. After the DNase treatment, the total RNA was isolated with QIAzol Lysis reagent. First strand cDNA was reverse transcribed from the total RNA with Superscript III reverse transcriptase (Invitrogen) and NotI-(dt)18 primer according to the manufacturer's protocol. TaqMan real-time PCR assay was performed containing first strand cDNA, TaqMan Gene Expression Master mix (Applied Biosystems, Foster City, CA), GTHβ, or lamprey EF1α primers and TaqMan probe. The PCR profile and the sequences of the primers and probe for GTHβ were previously described (31). The sequences of the primers and probe for EF1α are as follows: PM EF1A F primer (5′-CTGGCCACAGGGACTTCATC-3′), PM EF1A R primer (5′-ACCGGCCTCAAACTCACCTA-3′), and EF1A TaqMan probe (5′-FAM-ACATCGCAGGCTGACTGCGCC-TAMRA-3′). The expression level of GTHβ transcripts was normalized to that of EF1α (internal controls). The relative amounts of mRNA were represented as mean ± sem, and the statistical analysis was performed with one-way ANOVA followed by Fisher's PLSD. In all tests, the level of significance for different groups was P < 0.05. The detail method of quantitative PCR is described in Supplemental Materials and Methods.
Results
Gene organization and evolution of the precursors of LPXRFa peptides and PQRFa peptides in vertebrates
The cloned lamprey LPXRFa peptide precursor cDNA consisted of 992 nucleotides containing a short 5′ untranslated sequence of 90 bp, a single open reading frame of 477 bp, and a 3′ untranslated sequence of 425 bp with a poly(A) tail (Fig. 1). The translated lamprey LPXRFa peptide precursor polypeptide encoded three putative RFa peptide sequences with Gly residue as a C-terminal amidation signal followed by monobasic or dibasic endoproteolytic residues Arg or Lys (Fig. 1). We named the putative lamprey LPXRFa orthologous peptides as lamprey LPXRFa-1a, LPXRFa-1b, and LPXRFa- 2. Synteny analysis showed that a putative lamprey LPXRFa peptide gene was located near the CYCS gene in the Ensembl lamprey genome database, which was consistent with other vertebrates (Fig. 2A). Both the LPXRFa peptide gene and PQRFa peptide gene are located near the HOX clusters (Fig. 2, A and B). Because the HOX clusters are considered to have duplicated from a common ancestral gene during whole-genome duplication events through vertebrate evolution (44), we hypothesized that LPXRFa peptide and PQRFa peptide gene have diverged from a common ancestral gene through chromosome duplication (Fig. 2C). The lamprey LPXRFa peptide precursor showed a high sequence homology to other fish LPXRFa peptide precursors in the region encoding lamprey LPXRFa-1a and LPXRFa-1b (Fig. 2D, box B) and lamprey LPXRFa-2 (Fig. 2D, box C). The lamprey LPXRFa peptide precursor did not encode an RFa peptide in the corresponding region to fish LPXRFa-1 (Fig. 2D, box A). On the other hand, the precursors of PQRFa peptides encode two PQRFa peptides in fish and three in lamprey and hagfish (Fig. 2D).
Nucleotide sequence and deduced amino acid sequence of a cDNA encoding lamprey LPXRFa-1a, lamprey LPXRFa-1b, and LPXRFa-2. The sequences of these lamprey peptides are shown in bold. The lamprey LPXRFa peptide precursor cDNA consisted of 992 nucleotides containing a short 5′ untranslated sequence of 90 bp, a single open reading frame of 477 bp, and a 3′ untranslated sequence of 425 bp with a poly(A) tail. The open reading frame region began with a start codon at position 91 and terminated with a TGA stop codon at position 568. The cleavage site of the signal peptide of the deduced LPXRFa peptide precursor was predicted to be between the Val16-Ala17 bond. The signal peptide (16 aa) is underlined. The poly(A) adenylation signal AATAAA is also shown in bold. The nucleotide sequence is deposited in the DDBJ, European Molecular Biology Laboratory (EMBL), and GenBank sequence databases under accession no. AB661773.
Molecular evolution of LPXRFa peptide gene and PQRFa peptide gene. A, Synteny analysis of LPXRFa peptide gene loci in lamprey, human, chicken, X. tropicalis, and zebrafish chromosomes. Orthologous genes are linked by lines. The LPXRFa peptide genes are printed white on black. HOXA clusters are shown in gray box. B, Synteny analysis of PQRFa peptide gene loci in human, anole lizard, X. tropicalis, and zebrafish chromosomes. The PQRFa peptide genes are printed white on black. The HOXC clusters are shown in gray box. C, Proposed evolutionary history of LPXRFa and PQRFa peptide genes. LPXRFa and PQRFa peptide genes, originating from a common ancestral gene, may have evolved through chromosome duplication. D, Multiple amino acid sequence alignments of the precursors of LPXRFa peptides and PQRFa peptides. The conserved amino acids are shaded. The regions that encode LPXRFa or PQRFa peptides are boxed. NPY, Neuropeptide Y; MPP6, membrane protein, palmitoylated 6; RARG, retinoic acid receptor gamma; MYG, chromosome 12 open reading frame 10; TARBP2, TAR (HIV-1) RNA binding protein 2.
Phylogenetic analysis of the precursors of LPXRFa peptides and PQRFa peptides in vertebrates
By phylogenetic analyses, the lamprey LPXRFa peptide precursor grouped with the precursors of LPXRFa peptides but not with the precursors of PQRFa peptides in the phylogenetic tree (Fig. 3), although the lamprey precursor encoded PQRFa peptides (Fig. 1). In contrast, the previously identified lamprey PQRFa peptide precursor grouped with the precursors of PQRFa peptides (Fig. 3). The consensus tree showed high bootstrap percentage for all branches.
Unrooted phylogenetic tree of the precursor cDNA encoding lamprey LPXRFa peptide precursor, other identified or putative LPXRFa peptide precursors, and PQRFa peptide precursors in other vertebrates. Scale bar refers to a phylogenetic distance of 0.1-nucleotide substitutions per site. Numbers on the branches indicate bootstrap percentage after 1000 replications in constructing the tree. The position of lamprey LPXRFa precursor is boxed. The position of previously identified lamprey PQRFa peptide precursor is underlined.
Isolation and characterization of lamprey mature peptides
We employed immunoaffinity purification using the PQRFa antibody as described previously (34, 35). The fractions corresponding to the elution times of 44–46 min (Fig. 4Aa) and 46–48 min (Fig. 4Ab) showed intense immunoreactivities (Fig. 4A). These immunoreactive fractions were further independently subjected to another reversed-phase HPLC purification (Fig. 4, B and C). Three purified substances appeared to be eluted in separate single peaks (Fig. 4, B and C, c–e). Amino acid sequence analysis of the isolated substances detected the partial sequences of lamprey LPXRFa-2 from peak in Fig. 4Bc, LPXRFa-1a from peak in Fig. 4Bd, and LPXRFa-1b from peak in Fig. 4Ce (Supplemental Table 2). Because the C terminus of each peptide was expected to be Phe-NH2, we synthesized the three putative peptides with Phe-NH2 at their C termini and analyzed their behaviors by MALDI-TOF MS. Both native and synthetic peptides showed a similar molecular mass for each peptide (Fig. 4D). The isolated native peptides were determined as summarized in Fig. 4D.
HPLC profile of the retained material obtained by immunoaffinity purification using the antiserum against PQRFa peptide. A, The immunoreactive substances were eluted with 23.5–24.5% (a) and 24.5–25.5% (b) acetonitrile, indicated by the horizontal bars. B, HPLC profile of an immunoreactive substance eluted with 23.5–24.5% acetonitrile in A on a reverse-phase HPLC column. The immunoreactive substances eluted at 9 min (c) and 10 min (d), which were identified to be lamprey LPXRFa-2 and lamprey LPXRFa-1a by mass spectrometry, are indicated by arrows. C, HPLC profile of an immunoreactive substance eluted with 24.5–25.5% acetonitrile in A on a reverse-phase HPLC column. The immunoreactive substance eluted at 11 min (e), which was identified to be lamprey LPXRFa-1b by mass spectrometry, is indicated by an arrow. D, Behavior of the native and synthetic lamprey LPXRFa peptides on MALDI-TOF MS. The amino acid sequences of the identified peptides, and corresponding mass values of both native and synthetic peptides are indicated.
The C-terminal four amino acids of lamprey mature peptides, i.e. lamprey LPXRFa-1a, LPXRFa-1b, and LPXRFa-2, were identical to several LPXRFa peptides and to all PQRFa peptides, whereas the N-terminal sequences of these peptides were diverse (Table 1). Leucine, the typical fifth amino acid from the C-terminal end of LPXRFa peptides, was not conserved in lamprey mature peptides nor in grass puffer LPXRFa-2 (22) and medaka LPXRFa-2 (Tobari Y., T.Okamura, N.Kagawa, T.Osugi, K.Tsutsui, unpublished data). The C-terminal motif of PQRFa peptides was strictly conserved in human, bovine, lamprey, and hagfish (Table 1).
Comparison of the identified lamprey LPXRFa peptides with previously identified LPXRFa peptides and PQRFa peptides in other vertebrates
LPQRFamide or PQRFamide motifs are printed white on black. The C-terminal five amino acids are boxed in the dotted line. R-RFa, Rana RFamide; NPFF, neuropeptide FF; NPAF, neuropeptide AF.
* Putative sequences.
Comparison of the identified lamprey LPXRFa peptides with previously identified LPXRFa peptides and PQRFa peptides in other vertebrates
LPQRFamide or PQRFamide motifs are printed white on black. The C-terminal five amino acids are boxed in the dotted line. R-RFa, Rana RFamide; NPFF, neuropeptide FF; NPAF, neuropeptide AF.
* Putative sequences.
Expression of lamprey LPXRFa peptide precursor mRNA in various tissues
Lamprey LPXRFa peptide precursor mRNA was expressed in the brain and gonad but not in the pituitary of male and female lampreys (Fig. 5A). The single amplicon confirmed the specific target cDNA sequence (464 bp), which was not derived from genomic DNA, and there were not any splicing variants as the primers spanned all two putative intron/exon boundaries.
Expression of lamprey LPXRFa peptide in the brain. A, RT-PCR analyses of lamprey LPXRFa peptide precursor mRNA in brain, pituitary, and gonad of adult male and female lampreys. EF1α mRNA served as the positive control. Negative controls (NTC) containing no DNA template assured that there was no contamination in the PCR mixture. M, DNA size marker; ♂, male; ♀, female. B, Cellular localization of lamprey LPXRFa peptide precursor mRNA in the transverse brain sections identified by in situ hybridization. Lamprey LPXRFa peptide precursor mRNA was expressed in the rostral (a) and caudal (d) region of the nTPOC. The rectangular area in a or d is magnified in b or f. The sense RNA probe produced no hybridization signal as shown in c or f. Scale bars, 100 μm (a, b, d, and e) and 50 μm (c and f). C, Immunohistochemical staining of frontal brain sections using the antiserum against lamprey LPXRFa-2. Rectangular area in a or d is magnified in b or e. The immunoreaction of the hypothalamus was eliminated after preabsorption of the antibody with a saturating concentration of the peptide (c and f). Scale bars, 100 μm (a and d) and 40 μm (b, c, e, and f). ri, Infundibular recess.
Cellular localization of lamprey LPXRFa peptide precursor mRNA in the brain
An intense expression of lamprey LPXRFa peptide precursor mRNA was detected in the rostral region (Fig. 5B, a and b) and caudal region (Fig. 5B, d and e) of the bed nucleus of the tract of the postoptic commissure (nTPOC) in the hypothalamus. The control study using sense RNA probe resulted in a complete absence of staining (Fig. 5B, c and f).
Immunohistochemistry of lamprey LPXRFa-2 and GnRH-III in the brain
Lamprey LPXRFa-2 immunoreactive neurons were observed in the nTPOC in the hypothalamus (Fig. 5C). Lamprey GnRH-III immunoreactive neurons were densely distributed in the arc-shaped hypothalamic area extending from the nucleus preopticus to the nucleus commissure postopticae (Fig. 6B, a and b). Additional smaller numbers of GnRH-III positive neurons were found in the periventricular zone of the dorsal and ventral parts of the posterior hypothalamus. Appositions of lamprey LPXRFa-2 immunoreactive fibers were observed in close proximity to GnRH-III neurons in the nucleus preopticus-nucleus commissure postopticae (Fig. 6B, a and b). Only a few lamprey LPXRFa-2 immunoreactive fibers were observed in the neurohypophysis (Fig. 6B, c and d) compared with abundant lamprey GnRH-III immunoreactive fibers (Fig. 6B, e and f).
The projection of lamprey LPXRFa-2 and GnRH-III immunoreactive fibers in the hypothalamus. A, Schematic representation of the distribution of lamprey LPXRFa-2. Lamprey LPXRFa peptide-expressing cell bodies are shown by closed circles, whereas immunoreactive fibers are shown by thin lines. B, Immunohistochemistry of lamprey LPXRFa-2 and GnRH-III. Rectangular area in a, c, or e is magnified in b, d, or f. Double immunostaining of lamprey LPXRFa-2 and GnRH-III is shown in a and b. Lamprey LPXRFa-2 immunoreactive fibers are shown in brown, whereas lamprey GnRH-III immunoreactive cells and fibers are shown in red (a and b). Appositions of lamprey LPXRFa-2 immunoreactive fibers in close proximity of GnRH-III neurons were observed in b (arrows). Sparse lamprey LPXRFa-2 immunoreactive fibers shown in brown were observed in the neurohypophysis (d). Abundant lamprey GnRH-III immunoreactive fibers shown in brown were observed in the neurohypophysis (f). Scale bars, 1 mm (a, c, and e) and 40 μm (b, d, and f). AH, Adenohypophysis; ch, optic chiasma; CT, connective tissue; f. retr.; fasciculus retroflexus; Hab, habenula; LV, lateral ventricle; NCP, nucleus commissurae postopticae; NH, neurohypophysis; NPO, nucleus preopticus; OB, olfactory bulb; PC, posterior commissure; PI, pars intermedia; POR, preoptic recess; PPD, proximal pars distalis; Pr. H, primordium hippocampi; RPD, rostral pars distalis; IIIV, third ventricle.
Biological activities of lamprey mature peptides on GnRH concentration and GTHβ mRNA expression
Female lampreys treated with lamprey LPXRFa-2 at 100 μg/kg showed significant increases in lamprey brain GnRH-III (P < 0.05) protein concentrations compared with controls (Table 2). Lamprey LPXRFa-2 at 100 μg/kg also tended to increase lamprey brain GnRH-I (P < 0.1) protein concentrations compared with controls (Table 2). Lamprey LPXRFa-2 at 100 μg/kg (0.1 μg/g) resulted in a significant increase (P < 0.05) of mRNA expression of GTHβ (Table 2). There were no significant changes with any of the other treatments compared with controls.
Biological activities of lamprey LPXRFa peptides on GnRH concentration and GTHβ mRNA expression
| Peptides | lGnRH-I (ng/brain) | lGnRH-II (ng/brain) | lGnRH-III (ng/brain) | GTHβ/EF1α (mRNA expression) |
|---|---|---|---|---|
| Saline | 2.12 ± 0.41 | 0.40 ± 0.08 | 4.81 ± 1.38 | 0.27 ± 0.12 |
| Lamprey LPXRFa-1a (50 μg/kg) | 4.51 ± 1.72 | 0.45 ± 0.13 | 3.90 ± 0.92 | 0.31 ± 0.05 |
| Lamprey LPXRFa-1a (100 μg/kg) | 1.82 ± 0.58 | 0.60 ± 0.18 | 2.70 ± 0.72 | 0.18 ± 0.06 |
| Lamprey LPXRFa-1b (50 μg/kg) | 3.72 ± 1.18 | 0.79 ± 0.30 | 11.43 ± 4.11 | 0.15 ± 0.02 |
| Lamprey LPXRFa-1b (100 μg/kg) | 4.28 ± 1.42 | 0.40 ± 0.10 | 11.09 ± 4.91 | 0.14 ± 0.03 |
| Lamprey LPXRFa-2 (50 μg/kg) | 6.31 ± 2.44 | 0.88 ± 0.23 | 17.46 ± 6.87 | 0.44 ± 0.23 |
| Lamprey LPXRFa-2 (100 μg/kg) | 10.26 ± 3.75b | 0.79 ± 0.18 | 30.92 ± 14.26a | 0.65 ± 0.13a |
| Peptides | lGnRH-I (ng/brain) | lGnRH-II (ng/brain) | lGnRH-III (ng/brain) | GTHβ/EF1α (mRNA expression) |
|---|---|---|---|---|
| Saline | 2.12 ± 0.41 | 0.40 ± 0.08 | 4.81 ± 1.38 | 0.27 ± 0.12 |
| Lamprey LPXRFa-1a (50 μg/kg) | 4.51 ± 1.72 | 0.45 ± 0.13 | 3.90 ± 0.92 | 0.31 ± 0.05 |
| Lamprey LPXRFa-1a (100 μg/kg) | 1.82 ± 0.58 | 0.60 ± 0.18 | 2.70 ± 0.72 | 0.18 ± 0.06 |
| Lamprey LPXRFa-1b (50 μg/kg) | 3.72 ± 1.18 | 0.79 ± 0.30 | 11.43 ± 4.11 | 0.15 ± 0.02 |
| Lamprey LPXRFa-1b (100 μg/kg) | 4.28 ± 1.42 | 0.40 ± 0.10 | 11.09 ± 4.91 | 0.14 ± 0.03 |
| Lamprey LPXRFa-2 (50 μg/kg) | 6.31 ± 2.44 | 0.88 ± 0.23 | 17.46 ± 6.87 | 0.44 ± 0.23 |
| Lamprey LPXRFa-2 (100 μg/kg) | 10.26 ± 3.75b | 0.79 ± 0.18 | 30.92 ± 14.26a | 0.65 ± 0.13a |
Results are mean ± sem of five separate samples. lGnRH, Lamprey GnRH.
P < 0.05 vs. control group.
P < 0.1 vs. control group.
Biological activities of lamprey LPXRFa peptides on GnRH concentration and GTHβ mRNA expression
| Peptides | lGnRH-I (ng/brain) | lGnRH-II (ng/brain) | lGnRH-III (ng/brain) | GTHβ/EF1α (mRNA expression) |
|---|---|---|---|---|
| Saline | 2.12 ± 0.41 | 0.40 ± 0.08 | 4.81 ± 1.38 | 0.27 ± 0.12 |
| Lamprey LPXRFa-1a (50 μg/kg) | 4.51 ± 1.72 | 0.45 ± 0.13 | 3.90 ± 0.92 | 0.31 ± 0.05 |
| Lamprey LPXRFa-1a (100 μg/kg) | 1.82 ± 0.58 | 0.60 ± 0.18 | 2.70 ± 0.72 | 0.18 ± 0.06 |
| Lamprey LPXRFa-1b (50 μg/kg) | 3.72 ± 1.18 | 0.79 ± 0.30 | 11.43 ± 4.11 | 0.15 ± 0.02 |
| Lamprey LPXRFa-1b (100 μg/kg) | 4.28 ± 1.42 | 0.40 ± 0.10 | 11.09 ± 4.91 | 0.14 ± 0.03 |
| Lamprey LPXRFa-2 (50 μg/kg) | 6.31 ± 2.44 | 0.88 ± 0.23 | 17.46 ± 6.87 | 0.44 ± 0.23 |
| Lamprey LPXRFa-2 (100 μg/kg) | 10.26 ± 3.75b | 0.79 ± 0.18 | 30.92 ± 14.26a | 0.65 ± 0.13a |
| Peptides | lGnRH-I (ng/brain) | lGnRH-II (ng/brain) | lGnRH-III (ng/brain) | GTHβ/EF1α (mRNA expression) |
|---|---|---|---|---|
| Saline | 2.12 ± 0.41 | 0.40 ± 0.08 | 4.81 ± 1.38 | 0.27 ± 0.12 |
| Lamprey LPXRFa-1a (50 μg/kg) | 4.51 ± 1.72 | 0.45 ± 0.13 | 3.90 ± 0.92 | 0.31 ± 0.05 |
| Lamprey LPXRFa-1a (100 μg/kg) | 1.82 ± 0.58 | 0.60 ± 0.18 | 2.70 ± 0.72 | 0.18 ± 0.06 |
| Lamprey LPXRFa-1b (50 μg/kg) | 3.72 ± 1.18 | 0.79 ± 0.30 | 11.43 ± 4.11 | 0.15 ± 0.02 |
| Lamprey LPXRFa-1b (100 μg/kg) | 4.28 ± 1.42 | 0.40 ± 0.10 | 11.09 ± 4.91 | 0.14 ± 0.03 |
| Lamprey LPXRFa-2 (50 μg/kg) | 6.31 ± 2.44 | 0.88 ± 0.23 | 17.46 ± 6.87 | 0.44 ± 0.23 |
| Lamprey LPXRFa-2 (100 μg/kg) | 10.26 ± 3.75b | 0.79 ± 0.18 | 30.92 ± 14.26a | 0.65 ± 0.13a |
Results are mean ± sem of five separate samples. lGnRH, Lamprey GnRH.
P < 0.05 vs. control group.
P < 0.1 vs. control group.
Discussion
Reproduction is the driving force for successful evolution of all organisms. In vertebrates, the HPG axis is a very complex system. Although GnRH is considered the master control of reproduction, other neuropeptides, such as GnIH, have now been shown to exert major regulatory roles in the HPG axis (for reviews, see Refs. 1–3). The evolutionary history of this GTH-inhibitory system was unknown, because the existence of GnIH had not been investigated in agnathans. In this study, we identified an LPXRFa peptide gene encoding three peptides (LPXRFa-1a, LPXRFa-1b, and LPXRFa-2) from the brain of sea lamprey. Based on our analyses, we suggest that the LPXRFa peptide gene diverged from a common ancestral gene likely through gene duplication in the basal vertebrates. In addition, we also propose that one of the ancestral functions of the LPXRFa peptides was as a stimulatory neuropeptide of the HPG axis compared with the inhibitory function seen in later-evolved vertebrates (birds and mammals).
Identification of GnIH ortholog (LPXRFa peptide) gene in lamprey
In this study, we first cloned a GnIH ortholog (LPXRFa peptide) gene. The lamprey LPXRFa peptide precursor gene was located near the CYCS gene in the lamprey genome. It was reported that conserved synteny was observed around the LPXRFa peptide gene loci in the chromosomes of other vertebrates, where the CYCS gene was also located (21, 36). The loci of the LPXRFa peptide gene and CYC gene in the lamprey genome is consistent with those of other vertebrates. The deduced precursor polypeptide consisted of 159 amino acid residues, including the sequences of the three putative peptides, i.e. lamprey LPXRFa-1a, LPXRFa-1b, and LPXRFa-2. Phylogenetic analysis also showed that lamprey LPXRFa peptide precursor groups with the LPXRFa peptide precursors of vertebrates, whereas the previously identified lamprey PQRFa peptide precursor (34) groups with the PQRFa peptide precursors of vertebrates. High bootstrap values supported these phylogenetic positions. Based on these results, we conclude that the lamprey LPXRFa peptide precursor gene is the ortholog of LPXRFa peptide gene. This is the first reported identification of the LPXRFa peptide ortholog in the brain of agnathans.
Both LPXRFa peptide precursor gene and PQRFa peptide precursor gene were located near the HOX clusters in the chromosome (Fig. 2, A and B). The amphioxus, a member of the protochordates, contains a single HOX cluster (44, 45), whereas mammals contain four HOX clusters (HOXA, HOXB, HOXC, and HOXD) that are considered to have arisen through two rounds of whole-genome duplication events in early vertebrate evolution (44). Lampreys have at least two HOX clusters, although the exact organization of the HOX cluster is not yet clarified (46). These observations suggest that LPXRFa peptide and PQRFa peptide precursor genes have diverged from a common ancestral gene through gene duplication (Fig. 2C). It is possible that the common ancestor of these genes existed before the emergence of the vertebrate lineage.
Identification of mature LPXRFa peptides in lamprey
We identified three lamprey mature peptides as mature endogenous ligands. On the basis of amino acid sequence analysis and mass spectrometry, the primary amino acid structures of the isolated peptides were considered as follows: SGVGQGRSSKTLFQPQRFa (lamprey LPXRFa-1a), AALRSGVGQGRSSKTLFQPQRFa (lamprey LPXRFa-1b), and SEPFWHRTRPQRFa (lamprey LPXRFa-2). Because the 18-amino acid sequence from the C terminus of lamprey LPXRFa-1b (underlined) was identical to the amino acid sequence of lamprey LPXRFa-1a, lamprey LPXRFa-1a was considered to be a shorter form of lamprey LPXRFa-1b. The comparison of the amino acid sequences of the LPXRFa peptides showed that the C-terminal LPXRFa motif was conserved in mammals, birds, and amphibians, whereas it is diverse in lampreys and some fish, such as grass puffer (22) and medaka (Tobari Y., T.Okamura, N.Kagawa, T.Osugi, and K.Tsutsui, unpublished data), suggesting differences in structure-function activities (47).
Tissue distribution of lamprey LPXRFa peptide
Lamprey LPXRFa peptide precursor mRNA signal was detected in the nTPOC in the hypothalamus. As shown in the present and earlier studies, immunopositive neuronal GnRH perikarya are present in an arc-shaped population extending from ventral to dorsal preoptic areas, and fibers from these cells projected to the neurohypophysis via the preoptico-hypophyseal tract (30, 39, 48). Immunohistochemistry showed that lamprey LPXRFa-2 immunoreactive fibers form putative contact with lamprey GnRH-III neurons, suggesting functional interactions. This result is in line with the observation that appositions of LPXRFa peptide fibers are in close proximity to GnRH neurons in the hypothalamus of mammals and birds (10–12, 19). On the other hand, only few lamprey LPXRFa-2 immunoreactive fibers were observed in the neurohypophysis, where abundant lamprey GnRH-III immunoreactive fibers exist. These results suggest that lamprey LPXRFa peptide-2 acts on GnRH-III neurons rather than regulating pituitary function directly. Furthermore, the expression of LPXRFa peptide transcript in the gonads of adult male and female lamprey provides information that these peptides may play multiple roles in reproductive functions. A recent study from zebrafish also reported the ubiquitous expression pattern of LPXRFa peptide precursor mRNA as well as the receptors in the various tissues, including the gonads (21). It has also been suggested that LPXRFa peptides participate in reproductive functions of all hierarchies of HPG axis in mammalian and bird species (for reviews, see refs. 2, 3). Therefore, the gonadal expression of LPXRFa peptide precursor mRNA in both sexes of the lamprey imply conservation of reproductive functions of LPXRFa peptides in gonads throughout vertebrates.
Functional analysis of lamprey LPXRFa peptides
From our in vivo analysis, we showed that LPXRFa-2, one of the lamprey mature peptides, significantly stimulated the expression of GnRH-III protein in the hypothalamus and GTHβ mRNA in the pituitary. Adenohypophysial function is thought to be controlled by diffusion of neurohormones across the connective tissue barrier that separates the neurohypophysis and adenohypophysis, although there is little or no crossing of blood vessels or neurons between these regions in lampreys (49). Abundant lamprey GnRH-III immunoreactive fibers in the neurohypophysis suggest that lamprey GnRH-III acts on the pituitary by diffusion. The close proximity of lamprey LPXRFa-2 immunoreactive fibers to GnRH-III neurons suggests that lamprey LPXRFa-2 acts via GnRH-III neurons and subsequently stimulates the expression of GTHβ mRNA in the pituitary. Future in vitro studies using cultured pituitaries may provide further insights whether lamprey LPXRFa-2 can act directly on the pituitary. Lamprey GTHβ is considered to be an ancestral subunit of pituitary glycoprotein hormone that emerged before the divergence of pituitary glycoprotein hormone subunits, such as LHβ, FSHβ, and TSHβ (31, 33). The present study thus suggests conservation of the function of LPXRFa peptides on the regulation of pituitary glycoprotein hormone. The effect of ip injection of lamprey LPXRFa-2 on the expression of GnRH-III protein suggests that lamprey LPXRFa-2 crosses the biological barriers, such as the blood-brain barrier, to reach GnRH-III neurons. Although it is reported that lampreys have a blood-brain barrier (50, 51), the barrier of lampreys may be more permeable compared with that of mammals. Further studies are needed on the permeability of small peptides, such as LPXRFa peptides, through the blood-brain barrier in lampreys.
The effect of lamprey LPXRFa-2 on the expression of GTHβ mRNA was in contrast to the effects of LPXRFa peptides in mammals, birds, and some fish. In mammals and birds, numerous studies showed that LPXRFa peptides (GnIH and RFRP-3, a mammalian GnIH orthologous peptide) act to inhibit GTH release and synthesis (for reviews, see refs. 2, 3). In fish, zebrafish LPXRFa-3 inhibits LH release in goldfish (21), whereas gfLPXRFa peptides stimulate the mRNA expression of GTH in sockeye salmon (23) and grass puffer (22). It is interesting that LPXRFa peptides also stimulate the release of GH and PRL in amphibians (13, 14) and GH in fish (23). These studies suggest that although the GnIH gene family originates from agnathans, the function of neuropeptides diverged over time. The divergence of neuropeptide function is also seen in the study of kisspeptin between basal vertebrates and higher vertebrates. The recent study showed that kisspeptins, including putative lamprey kisspeptin-10 and kisspeptin-13, directly inhibited LH expression in the pituitary in eel (52), whereas kisspeptin is known as a stimulatory factor on the HPG axis in mammals (for a review, see Ref. 53). To understand the functional divergence of LPXRFa peptides and kisspeptins between basal vertebrates and jawed vertebrates, the identification of their receptors GPR147 and GPR54, as well as mature kisspeptins in lampreys, is needed. It is known that RF9 and peptide234 are the selective antagonists of GPR147 (54) and GPR54 (55) in mammals, respectively. The coinjection of these antagonists with endogenous ligands may provide further insights into the mode of action of LPXRFa peptides and kisspeptins in lampreys.
Conclusion
Our molecular and biochemical studies of lamprey LPXRFa peptides show that this gene likely diverged from a common ancestral gene and shares common features with later evolved vertebrates. Yet there is a difference in function. Our data provide evidence that GnIH emerged in agnathans as a stimulatory neuropeptide that later diverged to an inhibitory neuropeptide during course of evolution from basal vertebrate to later-evolved vertebrates (birds and mammals).
Acknowledgments
We thank Allisan Aquilina-Beck, Erin Allgood, Zach Hauser, Ciara Kazakis, Ciara Dimou, and Jeff Neale for excellent technical assistance. We also thank Wayne Decatur for the synteny analysis of LPXRFa and Dr. Kazuyoshi Ukena for valuable discussions.
This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan, 18107002, 22132004, and 22227002 (to K.T.). This work was also supported by the National Science Foundation Grant IOS-0849569, AES Hatch 332 (to S.A.S.) and by the University of New Hampshire Summer Undergraduate Reserach Fellowships (K.G. and D.D.). Partial funding was provided by the New Hampshire Agricultural Experiment Station (Scientific Contribution no. 2461).
Disclosure Summary: The authors have nothing to disclose.
T.O., D.D., and K.G. contributed equally to this work.
Abbreviations:
- CYCS
Cytochrome c
- DNase
deoxyribonuclease
- EF
elongation factor
- fGRP
frog growth hormone-releasing peptide
- gf
goldfish
- GnIH
GTH-inhibitory hormone
- GPR
G-protein coupled receptor
- GTH
gonadotropin
- HPG
hypothalamic-pituitary-gonadal
- LPXRFa peptide
LPXRFamide (X = L or Q) peptide
- MALDI-TOF MS
matrix assisted-laser desorption/ionization time-of-flight mass spectrometry
- nTPOC
bed nucleus of the tract of the postoptic commissure
- PLSD
protected least significant difference
- PM
Petromyzon marinus
- RFa peptide
RFamide peptide
- RFRP
RFamide-related peptide.
References
