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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. [ABSTRACT FROM AUTHOR]

In animal mitochondrial DNA inheritance, it remains largely unclear where the mitochondrial genetic bottleneck localizes and how it works in rewinding Müller’s ratchet. In a variety of different animals germ plasm mRNAs typically aggregate along with numerous mitochondria to form the mitochondrial cloud (MC) during oogenesis. The MC has been found to serve as messenger transport organizer for germ plasm mRNAs. Germ plasm RNAs in MC will specifically distribute to the primordial germ cells of the future embryo. It has been proposed that the MC might be the site where selected mitochondria accumulate for specific transmission to grandchildren but this idea received relatively little attention and the criterion by which mitochondria are selected remains unknown. Our recent results in zebrafish provided further evidence for selective mitochondria accumulation in the MC by showing that mitochondria with high-inner membrane potential tend to be recruited preferentially into the MC, and these mitochondria are transported along with germ plasm to the cortex of the vegetal pole. By analyzing the composition, behavior and functions of the MC, and in reviewing related literature, we found strong support for the proposition that the MC corresponds to the position and function of the mitochondrial genetic bottleneck. [ABSTRACT FROM AUTHOR]