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First published online October 7, 2005
Journal of Experimental Biology 208, 3851-3858 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01848

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Chloride turnover and ion-transporting activities of yolk-sac preparations (yolk balls) separated from Mozambique tilapia embryos and incubated in freshwater and seawater

Junya Hiroi^1,*, Hiroaki Miyazaki², Fumi Katoh³, Ritsuko Ohtani-Kaneko¹ and Toyoji Kaneko³

¹ Department of Anatomy, St Marianna University School of Medicine, Miyamae, Kawasaki 216-8511, Japan
² Department of Molecular Cell Biology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kamigyo, Kyoto 602-8566, Japan
³ Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo, Tokyo 113-8657, Japan

^* Author for correspondence (e-mail: j-hiroi{at}marianna-u.ac.jp)

Accepted 14 August 2005

We have recently established a unique in vitro experimental model for mitochondrion-rich cell (MRC) research, a `yolk-ball' incubation system, in which the yolk sac is separated from the embryonic body of Mozambique tilapia embryos and subjected to in vitro incubation. To evaluate the ion-transporting property of the yolk balls, we examined Cl^– content and turnover in yolk balls incubated in freshwater and seawater for 48 h, and distribution patterns of three ion transporters, Na⁺/K⁺-ATPase, Na⁺/K⁺/2Cl^– cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR), in MRCs in the yolk-sac membrane. The Cl^– turnover rate measured by whole-body influx of ³⁶Cl^– was about 60 times higher in yolk balls in seawater than in freshwater, while there was no essential difference in Cl^– content between them. Na⁺/K⁺-ATPase-immunoreactive MRCs were larger in yolk balls from seawater than yolk balls from freshwater. Distribution patterns of ion-transporting proteins allowed us to classify MRCs in freshwater yolk balls into three types: cells showing only basolateral Na⁺/K⁺-ATPase, cells showing basolateral Na⁺/K⁺-ATPase and apical NKCC, and cells showing basolateral Na⁺/K⁺-ATPase and basolateral NKCC. The seawater yolk balls, on the other hand, were characterized by the appearance of MRCs possessing basolateral Na⁺/K⁺-ATPase, basolateral NKCC and apical CFTR. Those seawater-type MRCs were considered to secrete Cl^– through the CFTR-positive apical opening to cope with diffusional Cl^– influx. These findings indicate that the yolk balls preserve the Cl^– transporting property of intact embryos, ensuring the propriety of the yolk ball as an in vitro experimental model for the yolk-sac membrane that contains MRCs.

Key words: yolk ball, mitochondrion-rich cell, tilapia, Oreochromis mossambicus, yolk-sac membrane, chloride, turnover