Cloning and tissue distribution of two GnRH receptors in pejerrey. Phylogenetics relationships by Leonardo G. GuiLGur & Gustavo M. SoMoza (1) AbsTRACT. - We obtained the full-length cloning and characterized two GnrH receptors (GnrHrs) from brain/pituitary tissue of pejerrey ish. Both presented the conserved features of this family of receptors. The GnRHR expression patterns showed that both receptors were widely expressed in the central nervous system and peripheral organs, suggesting that these GnrHrs could be mediating different functions besides reproduction. Phylogenetic analysis performed by alignment of the three extracellular loop sequences (ECLs) showed that these molecules segregate into two main clades correspond- ing to GnrHr types i and ii. Key words. - GnRH receptors - Pejerrey - Teleosts - Phylogeny. Cybium 2008, 32(2) suppl. : 43-44. (1) IIB-INTECH, CC 164 (B7130IWA), Chascomús, Argentina. [somoza@intech.gov.ar] Introduction The GnRH receptors (GnRHRs) are membrane receptors linked to guanine nucleotide binding regulatory proteins. They possess three main functional domains: a N-terminal segment, seven helical trans-membrane domains connected by hydrophilic intra- and extracellular loops and a C-termi- nal segment (Millar et al., 2004). Several GnRHR cDNAs from mammalian and non mammalian vertebrates have been cloned and characterized. As the GnRH ligand, multiple GnRHRs are expressed in an individual vertebrate species. in pejerrey (Odontesthes bonariensis) the presence of three GnRH forms have been demonstrated. Then the objective of this work was to look for GnrHrs in this species and ana- lyze their tissue distribution expression pattern. Also, as the phylogenetic study of GnrH receptors in vertebrates includ- ing mammals has given contradictory results (Kah et al. , 2007), we tried to perform a new study using only the three extracellular loop sequences (ECLs). Methods Adult pejerrey ish were obtained from the IIB-INTECH aquatic facilities. Different tissues were quickly dissected out and total RNA extracted. Different GnRHRs were char- acterized using different consensus primers and RACE-PCR. After the characterization of their full lengths, speciic prim- ers were designed and tested in RT-PCRs with different tis- sues. In addition, RT-PCRs of GnRHs precursors and a pejer- rey ß-actin were performed from each tissue using speciic primers for these molecules. Two phylogenetic analyses were performed. The irst one used a multisequence align- ment with all vertebrate GnRHR ORF protein sequences and the second was restricted to the three ECLs. Two trees were constructed by neighbor-joining method with the p-distance model (Mega soft.). Drosophila melanogaster and Aedes aegypti sequences were used as outgroups. Results and Discussion Two full-length GnRH receptor precursors were charac- terized from brain/pituitary tissue. The cDNAs encoding obGnRHR-I (DQ875596) and obGnRHR-II (DQ875595) included the conserved features of the vertebrate class A, rhodopsin-like family of G-protein coupled receptor super- family. obGnrHr-i presented wide distribution among tis- sues not directly related to reproduction (eyes, gills, kidney, olfactory mucosa, etc.) whereas obGnRHR-II was restricted to the central nervous system and the pituitary gland. The presence of GnRH-R, together with the ligands (data not shown) outside the reproductive axis suggests that they can also be involved in functions other than reproduction. The phylogenic analysis restricted to the ELC region showed that GnrHr segregated into two main clades in vertebrates (Fig. 1). Figure 1: Phylogenetic tree based on the three extracellular loop sequences of vertebrate GnRHRs.