Identification of His 5, Trp 7, Tyr 8-GnRH (chicken GnRH II) in amphibian brain

Abstract

GnRH immunoreactive and bioactive peptides in Xenopus laevis brain extract were investigated by high performance liquid chromatography (HPLC), radioimmunoassay with region-specific antisera raised against GnRH (mammalian), His 5, Trp 7, Tyr 8-GnRH (chicken II) and Tyr 3,Leu 5,Glu 6,Trp 7,Lys 8-GnRH (lamprey), and by assessment of biological activity. Two immunoreactive peptides eluted in the same positions as GnRH and His 5,Trp 7,Tyr 8-GnRH respectively in HPLC systems which were specifically designed to separate four known natural vertebrate GnRHs (mammalian, chicken I and II, salmon). The immunological properties of these two immunoreactive peaks, determined by relative interaction with three region-specific antisera raised against mammalian GnRH and two specific His 5,Trp 7,Tyr 8-GnRH antisera, were identical to those of GnRH and His 5,Trp 7,Tyr 8-GnRH. The immunoreactive peak co-eluting with His 5,Trp 7,Tyr 8-GnRH represented approximately one-third of the total brain GnRH. Both immunoreactive peaks stimulated luteinizing hormone (LH) release in a chicken dispersed pituitary cell bioassay, and the amounts of LH release stimulated by the two peaks were appropriate for these peaks being GnRH and His 5,Trp 7,Tyr 8-GnRH. A small hydrophobic peak with GnRH immunoreactivity eluted in the same position as Trp 7,Leu 8-GnRH (salmon), while Gln 8-GnRH (chicken I) and lamprey GnRH were not detected. Two additional rather hydrophilic peptides cross-reacted with a COOH-terminus-directed antiserum and had LH-releasing activity. LH-releasing activity was also detected in hydrophobic HPLC fractions. In summary, these data provide evidence for the presence of both GnRH and a second peptide with properties identical to His 5,Trp 7,Tyr 8-GnRH in X. laevis brain. The finding in amphibian brain of His 5,Trp 7,Tyr 8-GnRH, which has previously been described in species from bird, reptile and fish classes, lends support to our hypothesis that this widespread structural variant may represent an early-evolved and conserved form of GnRH.