Proteasomal interference prevents zona pellucida penetration and fertilization in mammals.
Sutovsky-P; Manandhar-G; McCauley-TC; Caamano-JN; Sutovsky-M; Thompson-WE; Day-BN
Biol Reprod 2004 Nov; 71(5):1625-1637
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.
Reproductive-system-disorders; Reproductive-system; Spermatogenesis; Spermatozoa; Fertility; Mammalian-cells; In-vitro-study; Immune-reaction; Immune-system; Mass-spectrometry; Microscopic-analysis
Department of Animal Science, University of Missouri-Columbia, S141 ASRC, 920 East Campus Dr., Columbia, MO 65211-5300
Biology of Reproduction
University of Missouri, Columbia, Missouri