Autophagy in Differentiation and Tissue Maintenance

Won-Hee Song, Peter Sutovsky, Song, Won-Hee, Sutovsky, Peter

A cell-free system using oocyte extracts is a valuable tool to study early events of animal fertilization and examine protein-protein interactions difficult to observe in whole cells. The process of postfertilization sperm mitophagy assures timely elimination of paternal, sperm-contributed mitochondria carrying potentially corrupted mitochondrial DNA (mtDNA). Cell-free systems would be especially advantageous for studying postfertilization sperm mitophagy as large amounts of oocyte extracts can be incubated with hundreds to thousands of spermatozoa in a single trial, while only one spermatozoon per zygote can be examined by whole-cell approaches. Since sperm mitophagy is species-specific, the abundantly available frog egg extracts commonly used for cell-free systems have to be replaced with isospecific mammalian oocyte extracts, which are difficult to obtain. Here we describe the protocol for a mammalian, porcine cell-free system consisting of permeabilized domestic boar spermatozoa co-incubated with cell extracts from porcine oocytes, suitable for studying the interactions of maternal, oocyte-derived mitophagy factors with paternal, sperm mitochondria.