Cell Tissue Res (1995) 279:485-493 Cell &Tissue Research 9 Springer-Verlag 1995 Characterization and localization of gonadotropin-releasing hormone in the brain and pituitary of the three-spined stickleback, Gasterosteus aculeatus Eva Andersson 1, 2, Jan Bogerd 1, Bertil Borg 2, Peter J. Sharp 3, Nancy M. Sherwood 4, Henk J. Th. Goos 1 1Department of Experimental Zoology, Utrecht University, Padualaan 8, NL-3584 CH Utrecht, The Netherlands 2 Department of Zoology, University of Stockholm, S- 106 91 Stockholm, Sweden 3 AFRC Institute of Animal Physiology and Genetics Research, Edinburgh Research Station, Roslin, Midlothian EH 25 9PS, UK 4 Department of Biology, University of Victoria, Victoria, B.C., Canada V8W 2Y2 Received: 20 June 1994 / Accepted: 8 August 1994 Abstract. Radioimmunoassay (RIA) studies on high- performance liquid chromatography (HPLC) fractions of brain extracts of the three-spined stickleback, Gas- terosteus aculeatus, provided evidence for at least two forms of gonadotropin-releasing hormone (GnRH). One form showed chromatographic and immunological prop- erties similar to that of synthetic salmon GnRH (sGnRH). A second, unidentified form of GnRH eluted in the same position as chicken GnRH I (cGnRH-I); however, it did not cross-react in a cGnRH-I RIA. Fur- thermore, it cannot be excluded that chicken GnRH II (cGnRH-II) and maybe one other unidentified form are present in the stickleback. The distribution of GnRH in the brain of breeding adult male sticklebacks was stud- ied by use of immunohistochemistry. Two antisera against sGnRH and antisera against mGnRH and cGnRH-II were applied on cryosections and visualized using the peroxidase-antiperoxidase method. Staining patterns were similar after incubations with all four anti- sera. Immunoreactive fibers were found in most parts of the brain. Three distinct groups of GnRH-immunoreac- tive perikarya were found in the nucleus olfactoretinalis, in the nucleus anterior periventricularis, and in the nu- cleus lateralis tuberis. Moreover, weakly stained cells occurred in a periventricular position in the midbrain. The proximal pars distalis of the pituitary, housing the gonadotropic cells, was richly innervated by GnRH-pos- itive fibers. In the pars intermedia and in the rostral pars distalis, immunoreactive fibers were absent. Key words: Gonadotropin-releasing hormone - Hypo- thalamus - Pituitary, pars distalis - High-performance liquid chromatography - Immunohistochemistry - Ra- dioimmunoassay - Stickleback, Gasterosteus aculeatus (Teleostei) Correspondence to: E. Andersson Introduction In teleosts, as in other vertebrates, the release of gonado- tropic hormone (GTH) is stimulated by gonadotropin-re- leasing hormone (GnRH) (for review, see Peter 1983). Sherwood et al. (1983) purified GnRH from brains of chum salmon, Oncorhynchus keta, and identified its pri- mary structure as Trp7-LeuS-GnRH (sGnRH). In addition to sGnRH, other GnRH forms have been found in tele- osts: chicken GnRH-II (cGnRH-II), mammalian GnRH (mGnRH), and catfish GnRH as well as yet unidentified forms (for review, see King and Millar 1992). GnRHs have been localized by immunohistochemis- try in the brain of a number of teleosts (e.g., goldfish, Carassius auratus, Kah et al. 1984, 1986; dwarf gou- rami, Colisa lalia, Oka and Ichikawa 1990; sea bass, Dicentrarchus labrax, Kah et al. 1991). Most consistent- ly, immunoreactive perikarya have been found in hypo- thalamic regions such as the nucleus preopticus and the nucleus lateralis tuberis. The neuroendocrine control of reproduction has been extensively studied in the three-spined stickleback, Gas- terosteus aculeatus, including studies on GnRH-binding in the pituitary (Andersson et al. 1989, 1992). However, the GnRH form(s) present in the stickleback brain have not been identified. The aim of the present study was to characterize the forms of GnRH in the stickleback and to localize GnRH-positive perikarya in the brain. To this end, the GnRH-system was analysed by means of HPLC, RIA, and immunohistochemistry. Materials and methods Experimental animals Adult three-s.p.ined sticklebacks, Gasterosteus aculeatus, were caught in the Oresund or in the Baltic Sea, Sweden. The fish were kept in brackish water under a photoperiod of 16 h light and 8 h dark (LD 16:8) at a temperature of 15-20 ~ C in 200-1200 1 aquaria with a sand-covered bottom. The water was aerated and filtered; there were no plants except some algae. The fish were fed daily with frozen mysids.