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

我们最近建立了一个独特的体外实验模型，为富卵黄细胞（MRC）的研究，一个'蛋黄球'孵化系统，其中卵黄囊分离的胚胎体的莫桑比克罗非鱼胚胎和进行体外培养。为了评估卵黄球的离子转运特性，我们检测了在淡水和海水中孵育48 h的卵黄球中Cl^-含量和周转，以及三种离子转运蛋白Na^+/K^+-ATP酶、Na^+/K^+/2Cl^-协同转运蛋白（NKCC）和囊性纤维化跨膜传导调节因子（CFTR）在卵黄囊膜中的分布模式。在<36>海水中，卵黄球中的Cl^-周转率比淡水中高60倍左右，但两者之间的Cl^-含量没有本质差异。Na^+/K^+-ATP酶免疫反应阳性的MRCs在海水卵黄球中比淡水卵黄球中大。离子转运蛋白的分布模式使我们能够将淡水卵黄球中的MRC分为三种类型：仅显示基底外侧Na^+/K^+-ATPase的细胞、显示基底外侧Na^+/K^+-ATPase和顶端NKCC的细胞以及显示基底外侧Na ^+/K^+-ATPase和基底外侧NKCC的细胞。另一方面，海水卵黄球的特征是出现了具有基底外侧Na^+/K^+-ATP酶、基底外侧NKCC和顶端CFTR的MRCs。这些海水型MRC被认为通过CFTR阳性根尖孔分泌Cl^-，以科普扩散性Cl^-内流。这些发现表明，卵黄球保留了完整胚胎的Cl^-转运特性，确保了卵黄球作为含有MRC的卵黄囊膜的体外实验模型的适当性。

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.