Confocal microscopy analysis of the activity of mitochondria contained within the ‘mitochondrial cloud’ during oogenesis in Xenopus laevis
We have used ratiometric confocal microscopy and three fluorescence techniques to study the distribution and activity of mitochondria in frog oocytes during the early stages of oogenesis. Mitochondria in frog oocytes during oogenesis were characterised by a high ratio in the ‘mitochondrial cloud’ and peri-nuclear region and a low ratio in mitochondria freely dispersed within the cytoplasm. We tested whether the high ratio visualised by the three techniques represented mitochondrial membrane potential by perturbing the mitochondrial membrane potential. Carbonyl cyanide p-(trifluoromethyl)phenylhydrazone (FCCP) caused the immediate destruction of the membrane potential, and consequent loss of fluorescence from the membrane-potential-sensitive confocal channel. In contrast, nigericin caused an increase in membrane potential represented by a steady increase in fluorescence ratio. These data demonstrate that mitochondrial activity can be measured during oogenesis in frog oocytes, and suggest that the mitochondrial cloud and perinuclear regions are characterised by highly active mitochondria.(Received April 17 01)
(Accepted May 15 01)
Key Words: ATP production; Mitochondrial membrane potential; Oocyte polarity; Oogenesis.
c1 All correspondence to: M. Wilding, Centre for Reproductive Biology, Clinica Villa del Sole, Via Manzoni, 15, 80126 Naples, Italy. Tel: +39 081 641689. Fax: +39 081 547251. E-mail: [email protected]