Ion-transporting ability and functional differentiation of chloride cells in the yolk-sac membrane of"yolk balls"

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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 ^<36>Cl^- 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 iontransporting 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.