At the transition from invertebrates to vertebrates, a novel GnRH-like peptide emerges in amphioxus

Abstract

Gonadotropin-releasing hormone (GnRH) is a critical reproductive regulator in vertebrates. Homologous peptides are also found in invertebrates, with a variety of characterized functions. In the amphioxus, an invertebrate that provides the best model for the transition to vertebrates, four GnRH receptors (GnRHRs) were previously described, but their native ligands were not identified. Using a more sensitive search methodology with hidden Markov models, we identified the first GnRH-like peptide confirmed in the amphioxus Branchiostoma floridae. This peptide specifically activated one of the four GnRHRs. Although the primary structure of this peptide was divergent from any previously isolated GnRH peptide, the minimal conserved residues found in all other GnRH superfamily members were retained. The peptide was immunolocalized in proximity of the central canal of the anterior nerve cord, a region where other neuropeptides and receptors have been found. Additionally, the amphioxus GnRH-like gene was positioned in a locus surrounded by syntenic homologs of the human GnRH paralogon. The amphioxus GnRH-like peptide, with its distinct primary structure, activated a receptor with equal potency to multiple ligands that span the GnRH superfamily.

Keywords: GnRH superfamily; adipokinetic hormone; amphioxus genome; corazonin; hormone evolution; receptor evolution.

Fig. 1.

Peptide, cDNA, and gene organization for GnRH-like hormone in amphioxus. (A) The cloned cDNA sequence from the beginning of the signal peptide to the stop codon is shown in black lowercase letters. The deduced amino acids are shown for the signal peptide in black capital letters; the GnRH sequence with a terminal amidation site (G) and dibasic cut site (RK) in green; and the GAP sequence in blue. The asterix (*) indicates the stop codon. (B) The organization of the amphioxus gnrh-like gene is compared with the human GNRH1 and limpet gnrh-like genes. The exon–intron data were obtained from the respective genome databases for amphioxus and human and from (Tsai and Zhang 2008) for limpet. The distances are drawn to scale according to the number of nucleotides. The signal peptides are shown by a black box, the GnRH region by green, GAP by blue, and the 5′- and 3′-untranslated regions in gray. The arrowhead (^▾) denotes the GnRH peptide coding region.

Fig. 2.

Alignment of GnRH superfamily peptide homologs from a variety of bilaterians. Putative full-length peptide sequences from representative species were manually aligned for GnRH, corazonin (Crz), and adipokinetic hormone (AKH), which are members of the GnRH superfamily of peptides. The vertebrate GnRH2, which is present throughout the vertebrates, is used as the standard for comparison of amino acid similarity among the peptides. The number of similar amino acids is shown on the right under the column marked Sim. The novel amphioxus GnRH is shown with its amino acid similarity to GnRH2 (five amino acids are similar to the GnRH2 standard, which has 10 amino acids). A second GnRH peptide (GnRHv, variant) that was identified in the amphioxus genome is shown below (this synthesized peptide did not activate any of the four known amphioxus GnRHRs).

Fig. 3.

Response of four amphioxus GnRHRs to human GnRH1, octopus, and amphioxus GnRH-like peptide, both oxidized and reduced. The percent induction of inositol phosphate one (IP1) accumulation compared with basal levels in COS-7 cells expressing amphioxus GnRHRs (R) is shown after induction with graded concentrations of peptide for 1 h. Cells were transfected with a vector containing (A) GnRHR1, (B) GnRHR2, (C) GnRHR3, or (D) GnRHR4 followed 48 h later by peptide addition. Each dose-response curve was generated by three independent experiments; error bars signify the mean ± standard error of means (SEM) of replicates. The concentration of peptide that stimulated the log EC₅₀ IP1 response is shown on the right. A dash (––) indicates that a response was not detected.

Fig. 4.

Immunolabeling of GnRH in the central nervous system (CNS) by an amphioxus-specific GnRH antiserum. (A) The transverse section through the head region of amphioxus Branchiostoma floridae shows the location of the brain-like anterior nerve cord (brown). A boxed area shows the site of GnRH labeling in the dorsal central canal. (B) Magnification of the boxed area shows GnRH label surrounding the open central canal (ventricle) or filling the canal (above) in the CNS. The cells surrounding the outside of the canal are identified by their nuclei, which are stained blue by hematoxylin. (C) A transverse section 0.2 mm posterior to (B), incubated with preimmune serum and showing the same region. (D) Cross section of amphioxus CNS approximately 0.7 mm posterior to that in (A). In the box and next to the arrowheads, labeling is observed near the ventral central canal. (E) An enlargement of the CNS from (D). A pair of black pigment cells is shown. (F) A transverse section adjacent to that of (D), labeled with preimmune serum instead of primary antibody, without specific labeling. (G) A transverse section adjacent to (F), labeled with preadsorbed primary antibody, without specific labeling. Typically, many cells (each identified by a blue nucleus) are clustered near the central canal as the CNS is not vascularized.

Fig. 5.

Synteny of amphioxus GnRH with human GnRH paralogons and limpet AKH. The amphioxus genome, version 2 (V2), scaffold (scaf.) 36 contains the GnRH gene (green) with its nearest neighbors of vasotocin (VT: red) and neuropeptide S (NPS: pink) on either side. Further along the scaffold are a number of selected genes (blue) that also show synteny with the human GnRH paralogon. Four human chromosomes (chr.) have GnRH genes (GnRH1 and GnRH2) that are functional or genes (“ghost” GnRH3 and GnRH4) that were lost (dotted lines around gene) during the evolution of the vertebrates, but the retained neighboring genes mark the location of the lost genes. An important comparison is between the conserved neighboring genes of GnRH and VT: amphioxus GnRH-VT, human GnRH2-VP/OXT (vasopressin/oxytocin), and the limpet AKH-VT. The association of amphioxus GnRH and NPS and the same in human GnRH3 and NPS is also shown. The neighboring genes are aligned for ease of comparison, but distances (shown below) and order may vary.

Fig. 6.

Phylogenetic tree of GnRH and VP receptor superfamily from vertebrates and invertebrates. Homologs of receptors used to generate a maximum likelihood tree are gonadotropin-releasing hormone (GnRHR: light green), adipokinetic hormone (AKHR: dark green), AKHR/Corazonin-related peptides (ACPR: dark green), corazonin (Crz: yellow), vasopressin family receptors (red) including oxytocin (OXTR), arginine vasopressin (AVPR), and vasotocin (VT), along with neuropeptides S (NPSR: pink), and crustacean cardioactive peptide (CCAPR: dark pink). The outgroup included a variety of neuropeptide receptors including those for tachykinin (TACR), somatostatin (SSTR), and galanin (GALR). The receptors are listed in supplementary table S1, Supplementary Material online. Nodes were compressed to represent animal lineages. The tree was prepared from a degapped alignment of receptors, using the conserved region of the seven-transmembrane domain including the intracellular and extracellular loops. RaxML 7.4.2 was used to generate the maximum likelihood tree topology, using the combined tree-search/fast bootstrap method under the PROTGAMMALG model (four discrete rate categories) with 1,000 fast bootstraps. Posterior probabilities are also listed at each node after the bootstrap value, from two converging chains generated in PhyloBayes MPI 1.4f under the same model constraints as the maximum likelihood tree. The two chains were sampled every 10 trees for 20,000 cycles (2,000 trees per chain) after an initial burn-in of 5,000 cycles was discarded.

Fig. 7.

Diagram showing the identification of GnRHR superfamily members and ligands. The presence of three families of receptors is shown in colored boxes for invertebrates and vertebrates from cnidarians to humans. The receptors (R) include chordate-type gonadotropin-releasing hormone (GnRHR), adipokinetic hormone (AKHR), and corazonin/invertebrate GnRH (CrzR/GnRHR). Also included are the corresponding peptides for the receptors. Red boxes indicate that receptors and peptides from respective groups have been isolated and are functional. Gray boxes indicate that gene predictions and/or transcripts have been identified for receptors and peptides, but they have not been characterized. The question marks in the hemichordate and echinoderm GnRHR and peptide boxes indicate that a group of GnRHR-like receptor and peptide sequences have been identified in Saccoglossus kowalevskii and Strongylocentrotus purpuratus (sea urchin), but their phylogenetic position is ambiguous. The question mark in the amphioxus GnRH box indicates that the peptide(s) activating GnRHR1 and 2 has been predicted but not identified.