In addition to vertebrate species

In addition to vertebrate species, GnRH has been identified in invertebrates, such as in the Protochordate Ciona intestinalis (Powell et al., 1996; Adams et al., 2003), the decapods mollusk Octopus vulgaris (Iwakoshi et al., 2002), the gastropod mollusk, the sea hare Aplysia californica (Zhang et al., 2008), the bivalve mollusk, the Yesso scallop Patinopecten yessoensis (Treen et al., 2012; Nagasawa et al., 2015) and the Pacific oyster Crassostrea gigas (Treen et al., 2012), and the crustacean, the red swamp crayfish Procambarus clarkii (Guan et al., 2014). The Annelida originally consists of three classes: Polychaeta, Oligochaeta, and Hirudinida. By recent phylogenomic analyses, Echiura, Pogonophora, and Sipuncula have been considered to belong to the Annelida (Struck et al., 2007, 2011; Ruggiero et al., 2015). Several invertebrate GnRH gene sequences in an annelid marine worm Capitella teleta (Polychaeta) and another annelid leech Helobdella robusta (Hirudinida) have been identified by the continual sequencing of genomes (Tsai and Zhang, 2008; Roch et al., 2011; Veenstra, 2011); the expected amino LY2606368 HCl sequences in marine worm GnRH (mwGnRH) and leech GnRH are pyroQAYHFSHGWFP-NH2 and pyroQSIHFSRSWQP-NH2, respectively. Currently, no information is available about the immunohistochemical localization of GnRH in annelids. In a pioneering immunohistochemical study on the distribution of several neuropeptides in the brain of the polychaeta Nereis diversicolor (Dhainaut-Courtois et al., 1985), GnRH was not detected by antibody raised against luteinizing hormone-releasing hormone (LHRH, mammalian form of GnRH), although some neuropeptides, such as corticotropin-releasing factor (CRF) and growth hormone releasing factor (GRF), were detected using vertebrate-type antibodies. The failure to detect GnRH by immunohistochemistry may be due to the use of the antibody raised against mammalian GnRH rather than one raised against mwGnRH. In the present study, since it is reasonable to speculate that annelids contain mwGnRH in the nervous system, we, for the first time, produced a rabbit polyclonal antibody raised against mwGnRH. Then, we examined the distribution of mwGnRH-immunoreactive (ir) cell bodies and fibers in the nerve ganglion of the clam worm Perinereis aibuhitensis (Annelida, Polychaeta) by immunohistochemistry.
Materials and methods
Results
Discussion We demonstrated the existence of mwGnRH immunoreactivities in the nerve ganglion of P. aibuhitensis by immunohistochemistry using a newly produced rabbit polyclonal antibody raised against mwGnRH. The specificity of the antibody was confirmed by dot blot assay. The antibody cross-reacted with mwGnRH, but not with other forms of GnRH. A number of mwGnRH-ir cell bodies were detected in nuclei 15–22, the caudal part of the cerebral ganglion. This region includes nucleus 15, which is supposed to be the synthesis site for neurohormones, such as cholecystokinin/gastrin, substance P, and β-melanocyte-stimulating hormone, which are involved in various physiological processes (Dhainaut-Courtois et al., 1985). The mwGnRH-ir fibers were mainly observed in the optic neuropil, and in the central neuropil region. Furthermore, a small number of mwGnRH-ir fibers were detected in the subpharyngeal ganglion and the ventral nerve cord. In contrast, in Nereis diversicolor (Annelida, Polychaeta), the immunoreactivities of CRF and GRF were stronger in the ventral nerve cord than in the cerebral ganglion (Dhainaut-Courtois et al., 1985). These results indicate that mwGnRH is synthesized in the cerebral ganglion and is transported through the subpharyngeal ganglion and the ventral nerve cord. The wide distribution of mwGnRH-ir fibers in Perinereis aibuhitensis also indicates that mwGnRH functions either as a neurotransmitter or neuromodulator. We previously examined the localization of GnRH-like peptides in the neural ganglia of the Pacific abalone Haliotis discus hannai (Mollusca, Gastropoda) and the chiton Acanthopleura japonica (Mollusca, Polyplacophora) by immunohistochemistry using LRH13 and aCII6 (Amano et al., 2010a, b). The cell bodies and fibers immunostained with these two antibodies were distinguished by dual-label immunohistochemistry, which suggested the existence of multiple GnRH-like peptides in these species. In this study, no immunoreactive cell bodies and fibers were detected by LRH13 or aCII6, which suggests that Perinereis aibuhitensis only contains mwGnRH, or that another form of GnRH exists in addition to mwGnRH, which does not cross-react with LRH13 and aCII6.