Origins of gonadotropin-releasing hormone (GnRH) in vertebrates: identification of a novel GnRH in a basal vertebrate, the sea lamprey

Abstract

We cloned a cDNA encoding a novel (GnRH), named lamprey GnRH-II, from the sea lamprey, a basal vertebrate. The deduced amino acid sequence of the newly identified lamprey GnRH-II is QHWSHGWFPG. The architecture of the precursor is similar to that reported for other GnRH precursors consisting of a signal peptide, decapeptide, a downstream processing site, and a GnRH-associated peptide; however, the gene for lamprey GnRH-II does not have introns in comparison with the gene organization for all other vertebrate GnRHs. Lamprey GnRH-II precursor transcript was widely expressed in a variety of tissues. In situ hybridization of the brain showed expression and localization of the transcript in the hypothalamus, medulla, and olfactory regions, whereas immunohistochemistry using a specific antiserum showed only GnRH-II cell bodies and processes in the preoptic nucleus/hypothalamus areas. Lamprey GnRH-II was shown to stimulate the hypothalamic-pituitary axis using in vivo and in vitro studies. Lamprey GnRH-II was also shown to activate the inositol phosphate signaling system in COS-7 cells transiently transfected with the lamprey GnRH receptor. These studies provide evidence for a novel lamprey GnRH that has a role as a third hypothalamic GnRH. In summary, the newly discovered lamprey GnRH-II offers a new paradigm of the origin of the vertebrate GnRH family. We hypothesize that due to a genome/gene duplication event, an ancestral gene gave rise to two lineages of GnRHs: the gnathostome GnRH and lamprey GnRH-II.

Figure 1

Putative lamprey GnRH-II transcript map. The putative prepro-lamprey GnRH-II nucleotide and deduced amino acid sequences. Regions corresponding to known GnRHs are shown; signal peptide is underlined, the lamprey GnRH-II decapeptide is boxed, the dibasic processing site is boxed in gray and the GnRH-associated peptide is dash-underlined. The stop codon is indicated by a hyphen.

Figure 2

Primary structure of GnRHs in vertebrates. There are currently 15 reported isoforms of GnRH in vertebrates. Yellow highlight, Amino acids changed, compared with mammalian GnRH; gray highlight, GnRHs occurring in the sea lamprey; orange highlight, single amino acid difference between dogfish, chicken-II, and lamprey GnRH.

Figure 3

Expression and analysis of lamprey GnRH-II. Expression of lamprey GnRH in several tissues is shown using RT-PCR (first panel); negative control, RNA that was not reverse transcribed (second panel). Lamprey actin was used to ensure quality of RNA (third panel) and actin without reverse transcription (RT; fourth panel). Results from PCR with genomic DNA template products (fifth panel) are the same size as those produced with RT-PCR, indicating the lack of introns. L, Liver; K, kidney; I, intestine; S, skeletal muscle; H, heart muscle; TH, thyroid; B, brain; T, testis; O, ovary; P, pituitary; E, eye; −, negative control.

Figure 4

Phylogenetic analysis of GnRH precursors. The neighbor-joining method with 1000 bootstrap replicates was used to produce this phylogenetic tree using the amino acid sequences of the prepro-GnRHs (signal peptide, decapeptide, dibasic cleavage site, and GAP) and is rooted with Aplysia-GnRH. The branch of lamprey GnRH-II is highlighted with a thick black line. Numbers indicate the percentage of bootstrap replicates in which the labeled branch was reproduced. m, Mammalian; gp, guinea pig; pj, pejerry; sb, seabream; wf, whitefish; cf., catfish; hr, herring; r, rana; s, salmon; c, chicken; lamprey; ap, Aplysia.

Figure 5

Evolution of vertebrate GnRH family of neuropeptides. This is a schematic diagram in which we hypothesize that lamprey GnRH-II evolved from a common ancestor of all Gnathostome forms of GnRH (black line). The gene duplication events that generated the different fish and tetrapod paralogous groups (D3, D4) took place within the Gnathostome lineage, after its divergence from the ancestral agnathans. The two other forms of lamprey GnRH (I and III, gray line) can be identified now as paralogous homologs of Gnathostome GnRH and lamprey GnRH II group, resulted from a duplication within the lamprey lineage (D2). This implies that there probably was a genome/gene duplication event, which gave rise to all forms of vertebrate GnRH affecting the common ancestor all of lamprey and Gnathostome isoforms (D1). The Gnathostome branch of the lamprey I and III orthologous group was probably lost during evolution (dotted gray line).

Figure 6

In situ hybridization of the adult lamprey brain. In situ hybridization of lamprey brain and sagittal tissue sections show distinct cell populations expressing lamprey GnRH-II and their relative locations within the lamprey brain. AS, Antisense probe; S, sense probe; V, ventriculi lateralis; SL, sulcus limitans. Scale bar, 100 μm.

Figure 7

IHC lamprey GnRH-II-like ir in the hypothalamus (hyp) of adult sea lampreys. Transverse (A) and sagittal (C) sections were stained with antilamprey GnRH-II, which was previously absorbed with a mixture of synthetic lamprey GnRH-I and -III. The areas outlined by the rectangle (A and C) are enlarged and shown in B, D, and E, respectively. Note lamprey GnRH-II-positive cell bodies in the preoptic nucleus (PON) and fibers in the neurohypophysis (NH). OC, Optic chiasm; PI, pars intermedia; POR, preoptic recess; PPD, proximal pars distalis; RPD, rostral pars distalis; III, third ventricle. Scale bars (A and C), 200 μm (A, ×50; C, ×40); B, D, and E, 20 μm (B, ×390; D, ×360; E, ×450).

Figure 8

In vitro ovarian and ovarian-pituitary cultures. Estradiol (picograms per milligram of tissue) after ovary treatments with HBSS (pH 7.0) with 25 mm HEPES (control), lamprey GnRH-I, -II, -III (10, 100, or 1000 ng/ml), coculture of pituitary and ovary treated with lamprey GnRH-I or -III (1000 ng/ml) and pituitary in HBSS (pH 7.0) with 25 mm HEPES. Lamprey GnRH-III-treated ovary had significantly higher estradiol (P < 0.05). The cocultures of pituitary and ovary with either lamprey GnRH-II or -III had significantly higher media estradiol concentrations, compared with controls (P < 0.05). *, Significance.

Figure 9

In vivo biological assay. Plasma estradiol (picograms per milliliter) following no injection (basal), injection with saline (control), lamprey GnRH-III (50 or 100 μg/kg), or lamprey GnRH-II (50 or 100 μg/kg). Lamprey GnRH-II (50 or 100 μg/kg) and lamprey GnRH-III (50 μg/kg) both increased plasma estradiol concentrations significantly (P < 0.05), compared with control. Lamprey GnRH-II (50 μg/kg) significantly increased plasma estradiol concentrations, compared with lamprey GnRH-III (P < 0.05). *, Significance when compared with control; **, significance when compared with control and l-GnRH-III.

Figure 10

IP assay lamprey GnRH dose-response curve. The lamprey GnRH receptor was shown to stimulate the IP signaling system in a dose-dependent manner, in transiently transfected COS7 cells. Lamprey GnRH-III stimulated IP accumulation at a significantly lower log EC₅₀ when compared with lamprey GnRH-II (P < 0.02) and lamprey GnRH-I (P < 0.001). Lamprey GnRH-II stimulated IP accumulation at a significantly lover log EC₅₀ when compared with lamprey GnRH-II (P < 0.02). Log EC₅₀ is shown as mean ± sem; n = 3 independent experiments.

Figure 11

GnRH gene structure of gnathostome and lamprey GnRH-I and –III. This figure depicts a schematic diagram of the genomic organization of the GnRH gene in vertebrates comprised of three introns and four exons. The lamprey GnRH-II gene consists of a single intron. For Ciona Ci-gnrh1 gene structure, Ci-gnrh1 does not have introns and consists of one exon with 5′ and 3′ flanking regions, in contrast to the three introns and four exon arrangement of the vertebrate GnRH gene.