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

我们最近建立了一种用于富线粒体细胞(MRC)研究的独特的体外实验模型，即从莫桑比克罗非鱼胚胎的胚体中分离卵黄囊并进行体外孵化的卵黄球孵化系统。为了评价卵黄球的离子转运性能，我们检测了在淡水和海水中孵育48h的卵黄球的氯离子含量和周转率，以及三种离子转运体Na+/K+-ATPase、Na+/K+/2Cl2-转运体(NKCC)和囊性纤维化跨膜电导调节因子(CFTR)在卵黄囊膜中的分布规律。海水中蛋黄球中的氯离子周转率约为淡水中的60倍，但两者的氯离子含量无本质差异。海水蛋黄球中Na+/K+-ATPase免疫反应阳性的MCs大于淡水蛋黄球。根据离子转运蛋白的分布模式，我们将淡水卵黄球中的细胞分为三种类型：仅基侧Na+/K+-ATPase的细胞，基侧Na+/K+-ATPase和顶端NKCC的细胞，以及基侧Na+/K+-ATPase和基侧NKCC的细胞。海水卵黄球则以基侧Na~+/K~+-ATPase、基侧NKCC和顶端CFTR为特征。这些海水型MMC被认为通过CFTR阳性的根尖开口分泌氯离子，以应对扩散的氯离子内流。这些结果表明，卵黄球保留了完整胚胎的氯离子转运特性，确保了卵黄球作为含有MRCs的卵黄囊膜的体外实验模型的合理性。

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.