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Proteasomal Interference Prevents Zona Pellucida Penetration and Fertilization in Mammals¹

Departments of Animal Science,³ Obstetrics and Gynecology,⁴ University of Missouri-Columbia, Columbia, Missouri 65211-5300 TMI Laboratories,⁵ Tucson, Arizona 85750 Department of Animal Sciences,⁶ University of Arizona, Tucson, Arizona 85721 Cooperative Reproductive Science Research Center,⁷ Morehouse School of Medicine, Atlanta, Georgia

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 ß-1i, ß-2i, -6, and ß-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 and ß 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.

¹ Supported by USDA New Investigator award 99-35203-11743 and USDA award 2002-02069 to P.S., who was also supported by NIH/NIOSH (award 7 R21 OH07324-02) and Food for the 21st Century Program of the University of Missouri-Columbia. B.N.D, J.N.C., and T.M. were, in part, supported by the collaborative animal research program Development of Biotechnology Tools for Improved Genetic and Reproductive Performance in Swine between the University of Missouri Department of Animal Sciences and Monsanto Animal Agriculture Group.

² Correspondence: Peter Sutovsky, University of Missouri-Columbia, S141 ASRC, 920 East Campus Dr., Columbia, MO 65211-5300. FAX: 573 884 5540; sutovskyp{at}missouri.edu

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