title: Early degradation of paternal mitochondria in domestic pig (Sus scrofa) is prevented by selective proteasomal inhibitors lactacystin and MG132.
abstract: Ubiquitin-dependent proteolysis has been implicated in the recognition and selective elimination of paternal mitochondria and mitochondrial DNA (mtDNA) after fertilization in mammals. Initial evidence suggests that this process is contributed to by lysosomal degradation of the ubiquitinated sperm mitochondrial membrane proteins. The present study examined the role of the proteasome-dependent protein degradation pathway of the ubiquitin system, as opposed to lysosomal proteolysis of the ubiquitinated proteins, in the regulation of sperm mitochondrion elimination after fertilization. Boar spermatozoa prelabeled with vital fluorescent mitochondrial probes MitoTracker were used to trace the degradation of paternal mitochondria after in vitro fertilization (IVF) of porcine oocytes. The degradation of sperm mitochondria in the cytoplasm of fertilized oocytes started very rapidly, i.e., within 12-20 h after insemination. Four stages of paternal mitochondrial degradation were distinguished, ranging from an intact mitochondrial sheath (type 1) to complete degradation (type 4). At 27-30 h postinsemination, 96% of zygotes contained the partially (type 3) or completely (type 4) degraded sperm mitochondria. Highly specific peptide inhibitors of the ubiquitin-proteasome pathway, lactacystin (10 and 100 microM) and MG132 (10 microM), efficiently blocked the degradation of the sperm mitochondria inside the fertilized egg when applied 6 h after insemination. Using 10 microM MG132, only 13.6% of fertilized oocytes screened 27-30 h after IVF displayed type 3 sperm mitochondria, and there was no incidence of type 4, completely degraded mitochondria. Although lactacystin is not a reversible agent, the effect of MG132 was fully reversible: zygotes transferred to regular culture medium after 24 h of culture with 10 microM MG132 resumed development and degraded sperm mitochondria within the next cell cycle. Surprisingly, penetration of the zona pellucida (ZP) was also inhibited by MG-132 and lactacystin when the inhibitors were added at insemination. Altogether, these data provide the first evidence of the participation of proteasomes in the control of mammalian mitochondrial inheritance and suggest a new role of the ubiquitin-proteasome pathway in mammalian fertilization.
authors.affiliation: Department of Animal Science, University of Missouri-Columbia, 65211, USA. sutovskyp@missouri.edu
authors.forename: Peter
authors.initials: P
authors.lastname: Sutovsky
authors.forename: Tod C
authors.initials: TC
authors.lastname: McCauley
authors.forename: Miriam
authors.initials: M
authors.lastname: Sutovsky
authors.forename: Billy N
authors.initials: BN
authors.lastname: Day
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000818:Animals; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D005260:Female; D005307:Fertilization in Vitro; D005455:Fluorescent Antibody Technique; D066298:In Vitro Techniques; D007976:Leupeptins / Q000494:pharmacology*; D008297:Male; D008854:Microscopy, Electron; D008928:Mitochondria / Q000187:drug effects* / Q000235:genetics / Q000648:ultrastructure; D008938:Mitosis / Q000502:physiology; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors; D010063:Ovum / Q000187:drug effects; D011247:Pregnancy; D046988:Proteasome Endopeptidase Complex; D013079:Sperm Maturation / Q000502:physiology; D013094:Spermatozoa / Q000187:drug effects* / Q000648:ultrastructure; D013552:Swine; D025801:Ubiquitin / Q000502:physiology; D015053:Zygote / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D009097:Multienzyme Complexes; D025801:Ubiquitin; C067713:lactacystin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D000111:Acetylcysteine