Biological and immunological characterization of multiple GnRH in an opisthobranch mollusk, Aplysia californica.

Gonadotropin-releasing hormone (GnRH) is a neurohormone central to the regulation of reproductive functions in vertebrates. Recently, several studies have reported the presence of GnRH immunoreactivity (IR) in a number of mollusks, suggesting that the distribution of GnRH may not be restricted to Phylum Chordata. In the present study, we extend our investigations to an opisthobranch mollusk, Aplysia californica, to characterize the source, chemical nature, and biological activity of molluscan GnRH-related molecules. Specific radioimmunoassays (RIAs) of various tissue extracts of Aplysia revealed that only ovotestis, hemocytes, and hemolymph contained significant amounts of GnRH that crossreacts with antisera raised against tunicate-I (tI) and mammalian (m) GnRH. Further RIAs and extractions revealed that the GnRH-IR in the hemolymph is biochemically and immunologically distinct from the GnRH-IR in the hemocytes and ovotestis. Using reverse-phase high-performance liquid chromatography coupled with RIAs, the GnRH-IR in the hemolymph was resolved into two major peaks. The first peak eluted earlier than most known forms of vertebrate GnRH, and the later peak coeluted with m, lamprey I, chicken II, and tI-GnRH. However, both peaks were broad and may contain a heterogeneous mixture of GnRH-IR. Immunocytochemical study showed that tI-GnRH-IR was present in the connective sheath surrounding the central nervous system, with a strong presence in what appeared to be vascular space, again suggesting the close association between Aplysia GnRH-IR and circulation. Finally, treatment of the neuroendocrine bag cells with chicken II GnRH significantly decreased the duration of the afterdischarge (AD, a characteristic pattern of electrical firing in bag cell neurons) and the number of action potentials fired during an AD, indicating the presence of a corresponding GnRH receptor in the Aplysia central nervous system. Overall, the results demonstrated the presence of multiple forms of GnRH-IR that crossreact with tI- and mGnRH antisera in A. californica and the ability of a vertebrate GnRH to alter Aplysia neural activity. Together, these data suggest that GnRH may be a factor released by the ovotestis and hemocytes into the circulation to alter neural functions. GnRH-IR produced by the latter may serve as a novel mediator of the neural and immune functions in Aplysia. Copyright 2000 Academic Press.