Zhang Yong-Zhong, Ouyang Ying-Chun, Hou Yi, Schatten Heide, Chen Da-Yuan, Sun Qing-Yuan
State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Datun Road, Chaoyang, Beijing 100101, China.
Dev Growth Differ. 2008 Mar;50(3):189-201. doi: 10.1111/j.1440-169X.2008.00988.x. Epub 2008 Feb 27.
The behavior of mitochondria during early oogenesis remains largely unknown in zebrafish. We used three mitochondrial probes (Mito Tracker Red CMXRos, Mito Tracker Green FM, and JC-1) to stain early zebrafish oocyte mitochondria, and confocal microscopy to analyze mitochondrial aggregation and distribution. By using fluorescence recovery after photobleaching (FRAP), we traced mitochondrial movement. The microtubule assembly inhibitor nocodazole and microfilament inhibitor cytochalasin B (CB) were used to analyze the role of microtubules and microfilaments on mitochondrial movement. By using the dual emission probe, JC-1, and oxidative phosphorylation uncoupler, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), we determined the distribution of active and inactive (low-active) mitochondria. Green/red fluorescence ratios of different sublocations in different oocyte groups stained by JC-1 were detected in merged (green and red) images. Our results showed that mitochondria exhibited a unique distribution pattern in early zebrafish oocytes. They tended to aggregate into large clusters in early stage I oocytes, but in a threadlike state in latter stage I oocytes. We detected a lower density mitochondrial area and a higher density mitochondrial area on opposite sides of the germinal vesicle. The green/red fluorescence ratios in different sublocations in normal oocytes were about 1:1. This implies that active mitochondria were distributed in all sublocations. FCCP treatment caused significant increases in the ratios. CB and nocodazole treatment caused an increase of the ratios in clusters and mitochondrial cloud, but not in dispersed areas. Mitochondria in different sublocations underwent fast dynamic movement. Inhibition or disruption of microtubules or microfilaments resulted in even faster mitochondrial free movement.
在斑马鱼中，早期卵子发生过程中线粒体的行为在很大程度上仍不清楚。我们使用了三种线粒体探针（Mito Tracker Red CMXRos、Mito Tracker Green FM和JC-1）对斑马鱼早期卵母细胞的线粒体进行染色，并利用共聚焦显微镜分析线粒体的聚集和分布情况。通过光漂白后荧光恢复（FRAP）技术，我们追踪了线粒体的运动。使用微管组装抑制剂诺考达唑和微丝抑制剂细胞松弛素B（CB）来分析微管和微丝在线粒体运动中的作用。通过使用双发射探针JC-1和氧化磷酸化解偶联剂羰基氰4-（三氟甲氧基）苯腙（FCCP），我们确定了活性和非活性（低活性）线粒体的分布。在合并（绿色和红色）图像中检测了JC-1染色的不同卵母细胞组中不同亚位置的绿/红荧光比率。我们的结果表明，线粒体在斑马鱼早期卵母细胞中呈现出独特的分布模式。它们在I期早期卵母细胞中倾向于聚集成大簇，但在I期后期卵母细胞中呈丝状状态。我们在生发泡的相对两侧检测到低密度线粒体区域和高密度线粒体区域。正常卵母细胞中不同亚位置的绿/红荧光比率约为1:1。这意味着活性线粒体分布在所有亚位置。FCCP处理导致比率显著增加。CB和诺考达唑处理导致簇和线粒体云区域的比率增加，但在分散区域没有增加。不同亚位置的线粒体进行快速动态运动。微管或微丝的抑制或破坏导致线粒体自由运动甚至更快。
