Rainbow Trout Eyed-Stage Embryos Demonstrate Melatonin Rhythms under Light–Dark Conditions as Measured by a Newly Developed Time-Resolved Fluoroimmunoassay Hideaki Yamada, * Hiroaki Chiba, * Masafumi Amano, * Masayuki Iigo, † and Munehico Iwata* ,1 * School of Fisheries Sciences, Kitasato University, Sanriku, Iwate 022-0101, Japan; and † Department of Anatomy, St. Marianna University, School of Medicine, Miyamae, Kawasaki, Kanagawa 216-8511, Japan Accepted August 31, 2001 Melatonin contents in eyed-stage embryos of rainbow trout ( Oncorhynchus mykiss) were measured by a newly developed, highly sensitive, and nonisotopic immunoas- say method:time-resolved fluoroimmunoassay (TR-FIA). The melatonin–bovine serum albumin (BSA) conjugate was immobilized by physical adsorption to the wells of microtiter plates. A competitive assay using two antibod- ies was performed among melatonin–BSA in the solid phase, samples, melatonin antibodies, and europium-la- beled secondary antibodies. The system showed a sensi- tivity of 0.6 pg/well. The embryos showed clear melato- nin rhythms under a 12L:12D photo cycle. Although the melatonin was detected in the egg yolk, the quantities were the same in both the light and dark phases. These results indicate that the melatonin secretion system has already functioned at least from the eyed-stage embryo in rainbow trout. © 2002 Elsevier Science (USA) INTRODUCTION It is well known that melatonin levels in the pineal organ, eye, and blood are markedly higher in the dark phase than those in the light phase of the light– dark photo cycle (Binkley, 1987). The melatonin rhythm in the pineal organ (Zachmann et al., 1992; Iigo et al., 1994) seems to be regulated by environmental factors such as photoperiod and temperature (Falco ´n et al., 1992; Iigo and Aida, 1995). Ocular melatonin rhythm is also reported in fishes (Falco ´ n and Collin, 1991; Cahill, 1996; Iigo et al., 1997; Gern et al., 1978; Zachmann et al., 1992); it is controlled by the light– dark photo cycle. Although melatonin rhythms have been observed in adult fishes and embryonic zebrafish (Kazimi and Ca- hill, 1999), nothing has been reported in the develop- ing embryos of salmonid fishes. Time-resolved fluoroimmunoassay (TR-FIA) tech- nique has been applied to measure various hormones (Hemmila ¨, 1994). The method has many advantages, including the use of nonisotopic tracers, relatively high sensitivity, and low background compared to conventional fluorescent materials such as fluorescein isothiocyanate (Yamada et al., 1997). This background results from measuring the specific fluorescence of europium using time resolution technology. In the current study, we developed a highly sensi- tive and nonisotopic immunoassay method (TR-FIA) for melatonin according to the antigen immobilization method (Yamada et al., 1997). The assay system was applied to obtain information on melatonin rhythms 1 To whom reprint requests should be addressed. General and Comparative Endocrinology 125, 41– 46 (2002) doi:10.1006/gcen.2001.7731, available online at http://www.idealibrary.com on 0016-6480/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved. 41