J Mol Evol (2007) 64:614–627 DOI: 10.1007/s00239-006-0125-8 Characterization of the cDNAs Encoding Three GnRH Forms in the Pejerrey Fish Odontesthes bonariensis (Atheriniformes) and the Evolution of GnRH Precursors Leonardo G. Guilgur,1 Guillermo Ortı´,2 Pablo H. Strobl-Mazzulla,1 Juan I. Fernandino,1 Leandro A. Miranda,1 Gustavo M. Somoza1 1 Laboratorio de Ictiofisiologı´a y Acuicultura, Instituto de Investigaciones Biotecnolo´gicas/Instituto Tecnolo´gico de Chascomu´s (CONICET-UNSAM), C.C. 164 (B7130IWA) Chascomu´s, Provincia de Buenos Aires, Argentina 2 School of Biological Sciences, University of Nebraska, Lincoln, NE 68588, USA Received: 23 May 2006 / Accepted: 12 February 2007 [Reviewing Editor: Dr. Axel Meyer] Abstract. Most vertebrates express two gonado- converge on this result. Although alternative scenar- tropin releasing hormone (GnRH) variants in brain ios could not be statistically rejected in this study due tissue but there is an increasing number of fish species to the relatively short size of the analyzed molecules, for which a third GnRH form has been detected. We this hypothesis also receives support from chromo- characterized the precursors (cDNAs) of all three somal studies of synteny around the GnRH genes in forms expressed in the brain of the pejerrey (silver- vertebrates. side) fish, Odontesthes bonariensis (Atheriniformes): type I (GnRH-I; 440 bp), type II (GnRH-II; 529 bp), Key words: Gonadotropin releasing hormone and type III (GnRH-III; 515 bp). The expression of (GnRH) — Phylogeny — mRNA expression — Brain these GnRHs precursors was also observed in — Reproduction — Teleosts — Pejerrey peripheral tissues related to reproduction (gonads), visual and chemical senses (eye and olfactory epi- thelium), and osmoregulation (gill), suggesting that in teleost fish and possibly other vertebrates GnRH mediates directly or indirectly many other functions Introduction besides reproduction. We also present a comprehen- sive phylogenetic analysis including representatives of Gonadotropin releasing hormone (GnRH) is a all chordate GnRH precursors characterized to date pivotal neuropeptide for development and repro- that supports the idea of two main paralogous GnRH duction of all vertebrates. GnRH is synthesized in lineages with different function. A ‘‘forebrain line- the hypothalamus and released into the pituitary age’’ separates evolutionarily from the ‘‘midbrain gland via the portal vascular system in tetrapods or lineage’’ as a result of an ancient duplication (ca. 600 via direct neuronal innervations in the case of tel- million years ago). A third, fish-only clade of GnRH eost fish. At the pituitary gland, the primary neu- genes seems to have originated before the divergence rohormonal function of GnRH is to stimulate the of fish and tetrapods but retained only in fish. Phy- synthesis and release of gonadotropins, which in logenetic analyses of GnRH precursors (DNA and turn, stimulate steroidogenesis and gonadal devel- protein sequences) under different optimality criteria opment. Comparative endocrinological studies have re- vealed 14 different GnRH variants in vertebrates, defined by the amino acid sequence of the active Correspondence to: Gustavo M. Somoza; email: somoza@intech. peptide (Lethimonier et al. 2004), but all variants gov.ar are decapeptides with residues 1, 4, 9, and 10 per- 615 fectly conserved. Fish harbor the highest diversity in the evolution of the GnRH gene family. Unfortu- GnRHs among vertebrates: 8 of the 14 described nately, direct phylogenetic analysis of GnRHs has GnRH variants have been characterized and/or been ineffective because these are short, conserved detected in teleosts and six are exclusive of this peptides with limited phylogenetic signal. Thus, lineage (see the Appendix for an account of the analyses have been based on genomic or cDNA se- intricate taxonomy and nomenclature of GnRH quences of the precursor (prepro-GnRH) that forms used in the endocrinological literature). The includes a signal peptide, the GnRH peptide itself, a early assumption that a single molecular form is conserved processing tripeptide, and the GnRH- expressed in all vertebrate brains has given way to associated peptide (GAP). Phylogenetic results to date the view that most vertebrates carry different GnRH suggest that vertebrate GnRH variants fall into three genes in their genomes and express more than one main clades: clade 1, with GnRH-I variants from GnRH variant in their brains (Fernald and White neurons located in the preoptic region with pituitary 1999). All fish species analyzed to date are known to function; clade 2, grouping all GnRH-II variants ex- express two or three different forms of GnRH. The pressed by midbrain neurons; and clade 3, with all most conserved form of GnRH (hereafter referred to fish-specific GnRH-III forms located either in neu- as type II or GnRH-II, first described in birds) is rons from the olfactory region and/or the telenc- found in Chondrichtyes and all other vertebrates, hephalon of fish (White et al. 1998; Okubo and Aida but all fish also express another GnRH variant re- 2001; Okubo et al. 1999; 2002). The first phylogenetic lated to the control of pituitary gonadotropes (type analysis of GnRHs (White et al. 1998), including few I or GnRH-I, first characterized in mammals). If a representatives of fish and tetrapods, concluded that a third form is present, this is a fish-specific GnRH gene duplication originating GnRH-I and GnRH-II variant, so far only described for teleosts (type III or forms must have occurred before the separation of GnRH-III). tetrapods from the ray-finned fish lineage. However, Studies in fish species expressing three GnRH the phylogenetic position of the GnRH-III lineage variants have shown that each variant exhibits remained uncertain in this and subsequent studies due differential distribution in the brain: the hypoph- to limited taxonomic sampling and no outgroup se- isotropic GnRH-I variant is mainly found in the quences from either chondrichthyans or agnathans preoptic-hypothalamic area, GnRH-II is expressed (but see Silver et al. 2004). In this context, two alter- by midbrain tegmentum (MT) neurons, and GnRH- native hypotheses have been considered: (i) the III by neurons located at the terminal nerve gan- GnRH-III lineage may have resulted from another glion (TNG) (e.g., Gothilf et al. 1996; Okuzawa ancient duplication predating the divergence of tet- et al. 1997; Fernald and White 1999; Vickers et al. rapods and ray-finned fish, and either has been lost in 2004). The distribution of these forms in different tetrapods later or has not yet been discovered; and (ii) clusters of cell bodies prompted some authors to the gene duplication leading to the GnRH-III lineage suggest the existence of three different GnRH sys- occurred recently, within the ray-finned fish lineage, tems with distinct physiological functions in fish explaining why these variants are restricted to fish (Parhar et al. 1998; Dubois et al. 2002). However, (Fig. 1; hypotheses A and B, respectively). Although except for the hypophysiotropic function of the different authors working in this field favor the former preoptic-hypothalamic GnRH-I form the physio- hypothesis (White et al. 1998; Okubo and Aida 2001; logical roles of other GnRHs are poorly known. Okubo et al. 1999, 2002), accumulating evidence Furthermore, the expression of these neuropeptides supporting more recent fish-specific whole-genomic in vertebrates seems not to be restricted to the duplication events (Amores et al. 1998; Christoffels central nervous system. Several reports have dem- et al. 2004; Hoegg et al. 2004; Van de Peer et al. 2003) onstrated GnRH expression outside the brain, in would favor the latter. A comprehensive phylogenetic tissues and organs such as the pituitary, skeletal analysis is necessary to fully understand the evolu- muscle, heart, liver, kidney, spleen, placenta, tionary diversification of the GnRH gene family. mammary gland, ovary, and testis (Kakar and Our laboratory is working with the pejerrey fish, Jennes 1995; White and Fernald 1998; Nabissi et al. Odontesthes bonariensis, as an experimental model for 2000; Uzbekova et al. 2001, 2002; Ikemoto and the study of the physiology of reproduction and sex Park 2003). Thus, GnRHs are likely to have di- determination and differentiation mechanisms. In the verse, and so far unknown, physiological functions present study we determine the cDNA sequences in addition to gonadotropin secretion. encoding all three GnRH forms in this species, doc- To understand the evolution of structural and ument their expression in different brain areas and functional diversification of these regulatory mole- extranervous tissues and organs, and also present cules, comparative studies of anatomical location and phylogenetic tests of the above hypotheses including taxonomic distribution of different GnRH variants representatives of all chordate GnRH precursors should be based on a phylogenetic framework to trace characterized to date. 616 Fig. 1. Alternative hypotheses for the evolution of the main groups of GnRH genes. A Two gene (genome) duplications (nodes 1R and 2R) occurred before the separation of fish and tetrapod lineages (shaded bars; estimated date of divergence, 450 MYA). Dashed line indicates loss of GnRH III genes in the lineage leading to tetrapods. The fish- specific genome duplication is not implied by this hypothesis. B Only one duplication (1R) occurred before the separation of fish and tetrapod lineages, and the origin of GnRH III genes follows the fish-specific genome duplication (3R). The diagram is a chronogram and estimated dates for nodes were obtained with a penalized likelihood method (see text and Table 2). Materials and Methods Amplification Primer; Table 1) as antisense primer was run under these conditions: 3 min at 94°C, followed by 35 cycles of 30 s at Animals and Sample Preparation 94°C, 30 s at 55°C, and 1 min at 72°C, ending with a 10-min extension step at 72°C. The PCR products obtained served as template for nested PCRs using the second corresponding sense Pejerrey fish were obtained from the stock maintained under primer (F2) in combination with the UAP-primer under similar natural environmental conditions in the outdoor ponds of the IIB- conditions.