Cell and Tissue Research

, Volume 341, Issue 2, pp 325–340 | Cite as

Interference with the 19S proteasomal regulatory complex subunit PSMD4 on the sperm surface inhibits sperm-zona pellucida penetration during porcine fertilization

  • Young-Joo Yi
  • Gaurishankar Manandhar
  • Miriam Sutovsky
  • Shawn W. Zimmerman
  • Věra Jonáková
  • Fred W. van Leeuwen
  • Richard Oko
  • Chang-Sik Park
  • Peter Sutovsky
Regular Article

Abstract

Proteolysis of ubiquitinated sperm and oocyte proteins by the 26S proteasome is necessary for the success of mammalian fertilization, including but not limited to acrosomal exocytosis and sperm-zona pellucida (ZP) penetration. The present study examined the role of PSMD4, an essential non-ATPase subunit of the proteasomal 19S regulatory complex responsible for proteasome-substrate recognition, in sperm-ZP penetration during porcine fertilization in vitro (IVF). Porcine sperm-ZP penetration, but not sperm-ZP binding, was blocked in the presence of a monoclonal anti-PSMD4 antibody during IVF. Inclusion in the fertilization medium of mutant ubiquitins (Ub+1 and Ub5+1), which are refractory to processing by the 19S regulatory complex and associated with Alzheimer’s disease, also inhibited fertilization. This observation suggested that subunit PSMD4 is exposed on the sperm acrosomal surface, a notion that was further supported by the binding of non-cell permeant, biotinylated proteasomal inhibitor ZL3VS to the sperm acrosome. Immunofluorescence localized PSMD4 in the sperm acrosome. Immunoprecipitation and proteomic analysis revealed that PSMD4 co-precipitated with porcine sperm-associated acrosin inhibitor (AI). Ubiquitinated species of AI were isolated from boar sperm extracts by affinity purification of ubiquitinated proteins using the recombinant UBA domain of p62 protein. Some proteasomes appeared to be anchored to the sperm head inner acrosomal membrane, as documented by co-fractionation studies. In conclusion, the 19S regulatory complex subunit PSMD4 is involved in the sperm-ZP penetration during fertilization. The recognition of substrates on the ZP by the 19S proteasomal regulatory complex is essential for the success of porcine/mammalian fertilization in vitro.

Keywords

Sperm Proteasome Fertilization Zona pellucida Acrosome Alzheimer’s Porcine 

Supplementary material

441_2010_988_MOESM1_ESM.jpg (120 kb)
Supplemental Data Fig. 1 (for web publication)

Visualization of acrosomal exocytosis and binding of the antibodies against subunit PSMD4 to the zona-bound boar sperm acrosomes during porcine IVF. In order to examine whether subunit PSMD4 contributes to sperm-ZP binding and acrosomal exocytosis shortly after sperm-oocyte mixing, ova were fertilized in the presence of different concentrations of mouse anti-PSMD4 antibody or without antibody, and fixed at 30 min after fertilization. The fixed ova, fertilized in the presence of anti-PSMD4 antibody, were incubated with a rabbit polyclonal antibody against sperm acrosomal tyrosine kinase YES, which detects intact acrosomes in ZP-bound boar spermatozoa (Sutovsky et al. 2004a), followed by incubation with a mixture of GAM-IgG-FITC and DAPI (DNA stain; blue). Panel (a) shows double-positive labeling at high magnification, panel (b) is a negative control for PSMD4 (with positive signal for YES), and panel (c) is a double positive labeling at low magnification (JPEG 120 kb).

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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Young-Joo Yi
    • 1
    • 2
    • 6
  • Gaurishankar Manandhar
    • 1
  • Miriam Sutovsky
    • 1
  • Shawn W. Zimmerman
    • 1
  • Věra Jonáková
    • 3
  • Fred W. van Leeuwen
    • 4
  • Richard Oko
    • 5
  • Chang-Sik Park
    • 2
  • Peter Sutovsky
    • 1
  1. 1.Division of Animal Sciences, and Department of Obstetrics, Gynecology & Women’s HealthUniversity of Missouri-ColumbiaColumbiaUSA
  2. 2.Research Center for Transgenic Cloned PigsChungnam National UniversityDaejeonSouth Korea
  3. 3.Laboratory of Diagnostic for Reproductive MedicineInstitute of Biotechnology Academy of Sciences of the Czech Republic v.v.i.Prague 4Czech Republic
  4. 4.School for Mental Health and Neuroscience, Department of NeuroscienceMaastricht UniversityMaastrichtThe Netherlands
  5. 5.Department of Anatomy and Cell BiologyQueen’s UniversityKingstonCanada
  6. 6.University of Missouri-ColumbiaColumbiaUSA

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