Hiroi Junya, Miyazaki Hiroaki, Katoh Fumi, Ohtani-Kaneko Ritsuko, Kaneko Toyoji
Department of Anatomy, St Marianna University School of Medicine, Miyamae, Kawasaki 216-8511, Japan.
J Exp Biol. 2005 Oct;208(Pt 20):3851-8. doi: 10.1242/jeb.01848.
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.
我们最近建立了一种独特的体外实验模型用于富含线粒体细胞（MRC）的研究，即“卵黄球”孵育系统，其中将莫桑比克罗非鱼胚胎的卵黄囊与胚胎体分离并进行体外孵育。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小时的卵黄球中的Cl⁻含量和周转率，以及卵黄囊膜中MRCs内三种离子转运蛋白（Na⁺/K⁺-ATP酶、Na⁺/K⁺/2Cl⁻共转运体（NKCC）和囊性纤维化跨膜传导调节因子（CFTR））的分布模式。通过³⁶Cl⁻全身内流测定的Cl⁻周转率在海水中的卵黄球中比在淡水中的卵黄球中高约60倍，而它们之间的Cl⁻含量没有本质差异。海水中卵黄球中Na⁺/K⁺-ATP酶免疫反应性MRCs比淡水中卵黄球中的更大。离子转运蛋白的分布模式使我们能够将淡水卵黄球中的MRCs分为三种类型：仅显示基底外侧Na⁺/K⁺-ATP酶的细胞、显示基底外侧Na⁺/K⁺-ATP酶和顶端NKCC的细胞以及显示基底外侧Na⁺/K⁺-ATP酶和基底外侧NKCC的细胞。另一方面，海水中的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。那些海水型MRCs被认为通过CFTR阳性的顶端开口分泌Cl⁻以应对扩散性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，确保了卵黄球作为包含MRCs 的卵黄囊膜的体外实验模型的适宜性。
