title: Proteasomal interference prevents zona pellucida penetration and fertilization in mammals.
abstract: The ubiquitin-proteasome pathway has been implicated in the penetration of ascidian vitelline envelope by the fertilizing spermatozoon (Sawada et al., Proc Natl Acad Sci U S A 2002; 99:1223-1228). The present study provides experimental evidence demonstrating proteasome involvement in the penetration of mammalian zona pellucida (ZP). Using porcine in vitro fertilization as a model, penetration of ZP was completely inhibited by specific proteasomal inhibitors MG-132 and lactacystin. Three commercial rabbit sera recognizing 20S proteasomal core subunits beta-1i, beta-2i, alpha-6, and beta-5 completely blocked fertilization at a very low concentration (i.e., diluted 1/2000 to 1/8000 in fertilization medium). Neither proteasome inhibitors nor antibodies had any effects on sperm-ZP binding and acrosome exocytosis in zona-enclosed oocytes or on fertilization rates in zona-free oocytes, which were highly polyspermic. Consistent with a possible role of ubiquitin-proteasome pathway in ZP penetration, ubiquitin and various alpha and beta type proteasomal subunits were detected in boar sperm acrosome by specific antibodies, immunoprecipitated and microsequenced by MALDI-TOF from boar sperm extracts. Antiubiquitin-immunoreactive substrates were detected on the outer face of ZP by epifluorescence microscopy. This study therefore provides strong evidence implicating the ubiquitin-proteasome pathway in mammalian fertilization and zona penetration. This finding opens a new line of acrosome/ZP research because further studies of the sperm acrosomal proteasome can provide new tools for the management of polyspermia during in vitro fertilization and identify new targets for contraceptive development.
authors.affiliation: Department of Animal Science, University of Missouri-Columbia, S141 ASRC, 920 East Campus Dr., Columbia, MO 65211-5300, USA. sutovskyp@missouri.edu
authors.forename: Peter
authors.initials: P
authors.lastname: Sutovsky
authors.forename: Gaurishankar
authors.initials: G
authors.lastname: Manandhar
authors.forename: Tod C
authors.initials: TC
authors.lastname: McCauley
authors.forename: Jose Nestor
authors.initials: JN
authors.lastname: Caamaño
authors.forename: Miriam
authors.initials: M
authors.lastname: Sutovsky
authors.forename: Winston E
authors.initials: WE
authors.lastname: Thompson
authors.forename: Billy N
authors.initials: BN
authors.lastname: Day
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000177:Acrosome / Q000378:metabolism; D020101:Acrosome Reaction / Q000187:drug effects; D000818:Animals; D005089:Exocytosis / Q000502:physiology; D005260:Female; D005306:Fertilization / Q000502:physiology*; D005307:Fertilization in Vitro; D007106:Immune Sera / Q000276:immunology / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D008297:Male; D046988:Proteasome Endopeptidase Complex / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D061988:Proteasome Inhibitors; D013084:Sperm-Ovum Interactions / Q000187:drug effects / Q000502:physiology*; D013094:Spermatozoa / Q000378:metabolism; D013552:Swine; D025801:Ubiquitin / Q000378:metabolism; D015044:Zona Pellucida / Q000378:metabolism / Q000502:physiology*
chemical_list: D007106:Immune Sera; D007976:Leupeptins; D061988:Proteasome Inhibitors; D025801:Ubiquitin; C067713:lactacystin; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D000111:Acetylcysteine