The accumulation of calcium into mitochondria during cellular calcium signalling represents a primary mechanism that regulates mitochondrial oxidative phosphorylation in response to the increased energy requirements of the cell so often associated with calcium signals. Furthermore, mitochondria may act as spatial buffers for calcium, and so alter the spatiotemporal patterning of calcium signals. In the oocyte, fertilisation triggers stereotypical patterns of calcium waves and oscillations upon which development of the early embryo depends. We have therefore sought evidence to define the influence of the calcium signals on mitochondrial energetics in the mammalian oocyte. This has required the simultaneous measurement and manipulation of [Ca²⁺]_c together with an assessment of mitochondrial redox state, an index of mitochondrial respiration and activity of the TCA cycle.

Oocytes are recovered from humanely killed MF1 mice as previously described (Halet et al. 2002). The isolated oocytes are mounted on a heated stage on a Zeiss 510 CLSM. Changes in [Ca²⁺]_c following fertilisation are measured using either rhod-2 or fura-red, loaded as the AM esters. Changes in mitochondrial function are measured as changes in NADH (ex. 351 nm, em. 405-485 nm) and/or flavoprotein autofluorescence (ex. 458 nm, em. > 505 nm), measured sequentially using the ‘multitracking’ facility of the Zeiss CLSM. We have in these experiments routinely seen oscillations in mitochondrial autofluorescence closely associated in time with [Ca²⁺]_c transients, strongly suggesting that [Ca²⁺]_c signals are transmitted to mitochondria and modulate mitochondrial metabolism. We will demonstrate a protocol for the simultaneous measurement of flavoprotein and NADH autofluorescence with [Ca²⁺]_c, together with the photolysis of caged InsP₃ or fertilisation to initiate [Ca²⁺]_c oscillations.

R.D. is supported by a Marie Curie Fellowship. J.C. and M.D. are funded by grants from the MRC and The Wellcome Trust.