Putative genes coding for pituitary adenylate cyclase-activating polypeptide and immunohistochemical localization of their mature peptides in the brain-pituitary axis of the inshore hagfish Eptatretus burgeri.

Hagfish and lampreys, which are both members of the class Agnatha, are widely considered to represent one of the most basal evolutionary lineages of vertebrates. Pituitary adenylate cyclase-activating polypeptide (PACAP) and growth hormone-releasing hormone (GHRH) are both members of the secretin/PACAP family. We searched the Ensembl database for putative PACAP and GHRH sequences in the inshore hagfish, Eptatretus burgeri. We identified three putative GHRH/PACAP precursors (hagfish-1/2/3) whose expression in various tissues, including the brain, was confirmed using RNA sequencing. The hagfish-1 precursor encodes both GHRH/PRP (PACAP-related peptide) and PACAP, whereas the hagfish-2 and -3 precursors encod only PACAP. Of the three putative hagfish PACAPs, PACAP-2, encoded by hagfish-2, showed relatively high identity (77.8%) and similarity (85.2%) with PACAP from other species. In contrast, hagfish GHRH/PRP encoded by hagfish-1 showed low identity (27.3%) and similarity (50.3%) with other GHRHs, and low identity (26.7%) and similarity (46.7%) with other PRPs. Phylogenetic analysis confirmed that the hagfish-2 sequence clustered within the same clade as lamprey PACAP. PACAP-like-immunoreactive (ir) cell bodies and dense punctate fibers have been detected in several brain regions, including the preoptic area, infundibular nucleus of the hypothalamus, midbrain tegmentum, and medulla oblongata. In the pituitary gland, PACAP-like-ir fibers were observed in the dorsal wall of the neurohypophysis. GHRH/PRP immunoreactivity was also observed in the medulla oblongata. The present study demonstrated that PACAP-like- and GHRH/PRP-like peptides are expressed in the brain of the inshore hagfish E. burgeri.