The GnRH system in zebrafish (Danio rerio): Developing a model system for functional studies of GnRH multiplicity

The presence of multiple forms of GnRH within the brain of a single species is a phenomenon that has been well characterized in lower vertebrates, particularly teleost fishes. Numerous studies in fish, amphibians and birds have demonstrated distinct ontogenies and different patterns of expression for each form of GnRH, indicating separate and unique roles in the reproductive process. Recently it has become clear that the paradigm of GnRH multiplicity extends to all vertebrates, including humans, although to date little is known about the functional significance of multiple GnRHs in a single species. Toward the development of a model to understand this, the present studies describe the characterization of the GnRH system in zebrafish. Full-length genes, including several kb of promoter sequence, were isolated for two forms of GnRH: sGnRH and the phylogenetically ubiquitous cGnRH-II. Localization studies in the adult brain show expression of cGnRH-II in the midbrain. sGnRH is found in the olfactory bulbs, anterior hypothalamus, and also the hindbrain. Using ELISAs, peptide levels in pooled pituitaries were found to be 8.9 and 2.4 pg/ml for sGnRH and cGnRH-II, respectively. The relative abundances in the pituitary and the localization in the brain demonstrate that zebrafish has two forms of GnRH, and the sGnRH and cGnRH-II forms correspond to the hypophysiotropic GnRH-I and midbrain GnRH-II, respectively. Developmental expression profiles of these two GnRHs were determined using whole-mount ISH and real-time quantitative RT-PCR assays. GnRH-II appears early in development, at 10-12 hpf, and arises from the midbrain. GnRH-I expression appears at 36 hpf in the nasal region, and subsequently migrates into the forebrain, as is seen in mammals. GnRH promoter transgenics using GFP and RFP reporter constructs confirm the differential patterns of expression for the two GnRH genes. These studies establish the necessary framework for future studies on the regulatory and functional aspects of multiple GnRHs in the zebrafish model system.