Interference with the 19S proteasomal regulatory complex subunit PSMD4 on the sperm surface inhibits sperm-zona pellucida penetration during porcine fertilization.
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.
Yi
Young-Joo
YJ
Division of Animal Sciences, and Department of Obstetrics, Gynecology & Women's Health, University of Missouri-Columbia, Columbia, MO, 65211, USA. yiyo@missouri.edu
Manandhar
Gaurishankar
G
Sutovsky
Miriam
M
Zimmerman
Shawn W
SW
Jonáková
Vera
V
van Leeuwen
Fred W
FW
Oko
Richard
R
Park
Chang-Sik
CS
Sutovsky
Peter
P
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D015153:Blotting, Western; D002352:Carrier Proteins / Q000276:immunology; D005307:Fertilization in Vitro / Q000187:drug effects; D008297:Male; D008969:Molecular Sequence Data; D046988:Proteasome Endopeptidase Complex / Q000276:immunology / Q000378:metabolism; D061988:Proteasome Inhibitors; D040901:Proteomics; D029607:Seminal Vesicle Secretory Proteins / Q000276:immunology; D013084:Sperm-Ovum Interactions / Q000187:drug effects; D013094:Spermatozoa / Q000201:enzymology*; D013552:Swine; D014359:Trypsin Inhibitor, Kazal Pancreatic / Q000276:immunology; D057149:Ubiquitinated Proteins / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D061988:Proteasome Inhibitors; D029607:Seminal Vesicle Secretory Proteins; C093236:Sp38 protein, Sus scrofa; D057149:Ubiquitinated Proteins; C087994:acrosin inhibitor, porcine; D014359:Trypsin Inhibitor, Kazal Pancreatic; D046988:Proteasome Endopeptidase Complex