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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Miyamoto K, Tsukiyama T, Yang Y, Li N, Minami N, Yamada M, Imai H (2009) Cell-free extracts from mammalian oocytes partially induce nuclear reprogramming in somatic cells. Biol Reprod 80(5):935–943. https://doi.org/10.1095/biolreprod.108.073676
Miyamoto K, Furusawa T, Ohnuki M, Goel S, Tokunaga T, Minami N, Yamada M, Ohsumi K, Imai H (2007) Reprogramming events of mammalian somatic cells induced by Xenopus laevis egg extracts. Mol Reprod Dev 74(10):1268–1277. https://doi.org/10.1002/mrd.20691
Sutovsky P, Simerly C, Hewitson L, Schatten G (1998) Assembly of nuclear pore complexes and annulate lamellae promotes normal pronuclear development in fertilized mammalian oocytes. J Cell Sci 111(Pt 19):2841–2854
Blow JJ, Laskey RA (1986) Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs. Cell 47(4):577–587
Zimmerman SW, Manandhar G, Yi YJ, Gupta SK, Sutovsky M, Odhiambo JF, Powell MD, Miller DJ, Sutovsky P (2011) Sperm proteasomes degrade sperm receptor on the egg zona pellucida during mammalian fertilization. PLoS One 6(2):e17256. https://doi.org/10.1371/journal.pone.0017256
Kaneda H, Hayashi J, Takahama S, Taya C, Lindahl KF, Yonekawa H (1995) Elimination of paternal mitochondrial DNA in intraspecific crosses during early mouse embryogenesis. Proc Natl Acad Sci U S A 92(10):4542–4546
Shitara H, Hayashi JI, Takahama S, Kaneda H, Yonekawa H (1998) Maternal inheritance of mouse mtDNA in interspecific hybrids: segregation of the leaked paternal mtDNA followed by the prevention of subsequent paternal leakage. Genetics 148(2):851–857
Abeydeera LR, Wang WH, Prather RS, Day BN (1998) Maturation in vitro of pig oocytes in protein-free culture media: fertilization and subsequent embryo development in vitro. Biol Reprod 58(5):1316–1320
Glikin GC, Ruberti I, Worcel A (1984) Chromatin assembly in Xenopus oocytes: in vitro studies. Cell 37(1):33–41
Ryoji M, Worcel A (1984) Chromatin assembly in Xenopus oocytes: in vivo studies. Cell 37(1):21–32
McLay DW, Clarke HJ (2003) Remodelling the paternal chromatin at fertilization in mammals. Reproduction 125(5):625–633
Perreault SD, Wolff RA, Zirkin BR (1984) The role of disulfide bond reduction during mammalian sperm nuclear decondensation in vivo. Dev Biol 101(1):160–167
Perreault SD (1992) Chromatin remodeling in mammalian zygotes. Mutat Res 296(1–2):43–55
Sutovsky P, Schatten G (1997) Depletion of glutathione during bovine oocyte maturation reversibly blocks the decondensation of the male pronucleus and pronuclear apposition during fertilization. Biol Reprod 56(6):1503–1512
Sutovsky P, Tengowski MW, Navara CS, Zoran SS, Schatten G (1997) Mitochondrial sheath movement and detachment in mammalian, but not nonmammalian, sperm induced by disulfide bond reduction. Mol Reprod Dev 47(1):79–86. https://doi.org/10.1002/(SICI)1098-2795(199705)47:1<79::AID-MRD11>3.0.CO;2-V
Bedford JM, Calvin HI (1974) Changes in -S-S- linked structures of the sperm tail during epididymal maturation, with comparative observations in sub-mammalian species. J Exp Zool 187(2):181–204. https://doi.org/10.1002/jez.1401870202
Sutovsky P (2004) Visualization of sperm accessory structures in the mammalian spermatids, spermatozoa, and zygotes by immunofluorescence, confocal, and immunoelectron microscopy. Methods Mol Biol 253:59–77. https://doi.org/10.1385/1-59259-744-0:059
Song WH, Yi YJ, Sutovsky M, Meyers S, Sutovsky P (2016) Autophagy and ubiquitin-proteasome system contribute to sperm mitophagy after mammalian fertilization. Proc Natl Acad Sci U S A 113(36):E5261–E5270. https://doi.org/10.1073/pnas.1605844113
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media New York
About this protocol
Cite this protocol
Song, WH., Sutovsky, P. (2018). Porcine Cell-Free System to Study Mammalian Sperm Mitophagy. In: Turksen, K. (eds) Autophagy in Differentiation and Tissue Maintenance. Methods in Molecular Biology, vol 1854. Humana Press, New York, NY. https://doi.org/10.1007/7651_2018_158
Download citation
DOI: https://doi.org/10.1007/7651_2018_158
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8747-4
Online ISBN: 978-1-4939-8748-1
eBook Packages: Springer Protocols