ATP Imaging in Xenopus laevis Oocytes

Abstract

Adenosine 5′-triphosphate (ATP) is a major energy currency for various chemical reactions in living cells. In spite of its signifi cant roles, the local ATP timecourse in Xenopus oocyte/egg has not been studied fully yet. Therefore, our goal is to understand the molecular mechanisms of maturation and fertilization by analyzing ATP in oocytes and eggs. A fl uorescence resonance energy transfer (FRET)-based ATP indicator, named ATeam, has been developed and enabled ATP imaging. We are applying this latest experimental technique to observe local ATP in Xenopus oocytes and eggs during maturation and fertilization. First, we used full-grown oocytes to set up the experimental conditions. To observe ATeam fl uorescence in Xenopus oocytes, the translucent oocytes were prepared after injecting ATeam protein into the vegetal hemisphere. Then they were observed under fl uorescence microscopy, and FRET was measured by analyzing their images using software. ATeam displayed strong FRET with low background. More importantly, FRET signal of ATeam consistently increased in response to the addition of ATP, suggesting that ATeam works in the translucent oocytes. Using this Xenopus ATP imaging system, ATP distribution is investigated not only in oocytes during maturation but also in eggs during fertilization.

Focused Proteomics on Egg Membrane Microdomains to Elucidate the Cellular and Molecular Mechanisms of Fertilization in the African Clawed Frog Xenopus laevis

“…Thus, this ongoing project will explore the presence of MDs-mitochondria interactions at fertilization and their physiological relevance to the metabolism and viability of developing embryos. In this connection, we have started to analyze quantitative and spatiotemporal regulation of ATP in oocytes and eggs during oocyte maturation and fertilization (Ijiri et al 2014 ).…”