Inhibition of 19S proteasomal regulatory complex subunit PSMD8 increases polyspermy during porcine fertilization in vitro

Yi, Young-Joo; Manandhar, Gaurishankar; Sutovsky, Miriam; Jonáková, Věra; Park, Chang-Sik; Sutovsky, Peter

doi:10.1016/j.jri.2009.11.002

« Previous Next »Journal of Reproductive Immunology
Volume 84, Issue 2 , Pages 154-163, March 2010

Inhibition of 19S proteasomal regulatory complex subunit PSMD8 increases polyspermy during porcine fertilization in vitro

Young-Joo Yi
- Division of Animal Sciences, and Department of Obstetrics and Gynecology, University of Missouri, Columbia, MO 65211, USA
- Research Center for Transgenic Cloned Pigs, Chungnam National University, Daejeon 305-764, South Korea
Gaurishankar Manandhar
- Division of Animal Sciences, and Department of Obstetrics and Gynecology, University of Missouri, Columbia, MO 65211, USA
Miriam Sutovsky
- Division of Animal Sciences, and Department of Obstetrics and Gynecology, University of Missouri, Columbia, MO 65211, USA
Věra Jonáková
- Laboratory of Diagnostic for Reproductive Medicine, Institute of Biotechnology Academy of Sciences of the Czech Republic v.v.i., 142 20 Prague 4, Czech Republic
Chang-Sik Park
- Research Center for Transgenic Cloned Pigs, Chungnam National University, Daejeon 305-764, South Korea
Peter Sutovsky
- Division of Animal Sciences, and Department of Obstetrics and Gynecology, University of Missouri, Columbia, MO 65211, USA
- Corresponding author at: University of Missouri-Columbia, S141 ASRC, 920 East Campus Drive, Columbia, MO 65211-5300, USA. Tel.: +1 573 882 3329; fax: +1 573 884 5540.

Received 16 June 2009; received in revised form 20 August 2009; accepted 5 November 2009. published online 27 November 2009.

Abstract

The 26S proteoasome is a multi-subunit protease specific to ubiquitinated substrate proteins. It is composed of a 20S proteasomal core with substrate degradation activity, and a 19S regulatory complex that acts in substrate recognition, deubiquitination, priming and transport to the 20S core. Inhibition of proteolytic activities associated with the sperm acrosome-borne 20S core prevents fertilization in mammals, ascidians and echinoderms. Less is known about the function of the proteasomal 19S complex during fertilization. The present study examined the role of PSMD8, an essential non-ATPase subunit of the 19S complex, in sperm-ZP penetration during porcine fertilization in vitro (IVF). Immunofluorescence localized PSMD8 to the outer acrosomal membrane, acrosomal matrix and the inner acrosomal membrane. Colloidal gold transmission electron microscopy detected PSMD8 on the surface of vesicles in the acrosomal shroud, formed as a result of zona pellucida-induced acrosomal exocytosis. Contrary to the inhibition of fertilization by blocking of the 20S core activities, fertilization and polyspermy rates were increased by adding anti-PSMD8 antibody to fertilization medium. This observation is consistent with a possible role of PSMD8 in substrate deubiquitination, a process which when blocked, may actually accelerate substrate proteolysis by the 26S proteasome. Subunit PSMD8 co-immunoprecipitated with acrosomal surface-associated spermadhesin AQN1. This association indicates that the sperm acrosome-borne proteasomes become exposed onto the sperm surface following the acrosomal exocytosis. Since immunological blocking of subunit PSMD8 increases the rate of polyspermy during porcine fertilization, the activity of the 19S complex may be a rate-limiting factor contributing to anti-polyspermy defense during porcine fertilization.

Keywords: Fertilization, Proteasome, Sperm, Acrosome, Zona pellucida