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pp 1-11 | Cite as

Porcine Cell-Free System to Study Mammalian Sperm Mitophagy

  • Won-Hee Song
  • Peter Sutovsky
Protocol
Part of the Methods in Molecular Biology book series

Abstract

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.

Keywords

Autophagy Cell-free system Extract Immunostaining Mitochondria Oocyte Porcine Sperm 

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Copyright information

© Springer Science+Business Media New York 2018

Authors and Affiliations

  1. 1.Division of Animal SciencesUniversity of MissouriColumbiaUSA
  2. 2.Department of Obstetrics, Gynecology and Women’s HealthUniversity of MissouriColumbiaUSA
  3. 3.Department of Animal Science and TechnologyChung-Ang UniversityAnseongSouth Korea

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