Mitochondrial behavior during oogenesis in zebrafish: a confocal microscopy analysis.
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.
Zhang
Yong-Zhong
YZ
State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Datun Road, Chaoyang, Beijing 100101, China.
Ouyang
Ying-Chun
YC
Hou
Yi
Y
Schatten
Heide
H
Chen
Da-Yuan
DY
Sun
Qing-Yuan
QY
D008841:Actin Cytoskeleton / Q000187:drug effects / Q000502:physiology; D000818:Animals; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D003571:Cytochalasin B / Q000494:pharmacology; D005260:Female; D036681:Fluorescence Recovery After Photobleaching; D005456:Fluorescent Dyes / Q000032:analysis; D018613:Microscopy, Confocal; D008870:Microtubules / Q000187:drug effects / Q000502:physiology; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure*; D009068:Movement; D015739:Nocodazole / Q000494:pharmacology; D009865:Oocytes / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure*; D009866:Oogenesis / Q000502:physiology*; D010085:Oxidative Phosphorylation / Q000187:drug effects; D014475:Uncoupling Agents / Q000494:pharmacology; D015027:Zebrafish / Q000502:physiology*
D005456:Fluorescent Dyes; D014475:Uncoupling Agents; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D003571:Cytochalasin B; D015739:Nocodazole