Submitted March 5, 2007
Returned for revision April 6, 2007
Accepted July 1, 2007
Gamete Biology
Ubiquitin C-Terminal Hydrolase-Activity Is Involved in Sperm Acrosomal Function and Anti-Polyspermy Defense During Porcine Fertilization
Young-Joo Yi ,
Gaurishankar Mananadhar ,
Miriam Sutovsky ,
Rongfeng Li ,
Vera Jonáková ,
Richard Oko ,
Chang-Sik Park ,
R. S. Prather ,
and
Peter Sutovsky *
* To whom correspondence should be addressed. E-mail: sutovskyp{at}missouri.edu.
Abstract
The 26S proteasome, a multi-subunit protease specific for substrate-proteins postranslationally modified by ubiquitination, has been implicated in the acrosomal function and sperm-zona pellucida (ZP) penetration during mammalian fertilization. Ubiquitin C-terminal hydrolases (UCHs) are responsible for the removal of polyubiquitin chains during substrate priming for proteasomal proteolysis. The inhibition of deubiquitination increases the rate of proteasomal proteolysis. Consequently, we have hypothesized that inhibition of sperm acrosome-borne UCHs will increase the rate of sperm-ZP penetration and polyspermy during porcine fertilization in vitro (IVF). Ubiquitin aldehyde (UA), a specific, non-permeant UCH-inhibitor, significantly (p<0.05) increased polyspermy during porcine IVF and reduced (p<0.05) enzymatic UCH-activity measured in motile boar spermatozoa by using a specific fluorometric UCH-substrate, ubiquitin-AMC. Antibodies against two-closely related UCHs, UCHL1 and UCHL3, detected these UCHs in the oocyte cortex and on the sperm acrosome, respectively, and increased the rate of polyspermy during IVF, consistent with the UA-induced polyspermy surge. In the oocyte, UCHL3 was primarily associated with meiotic spindle. Sperm-borne UCHL3 was localized to acrosomal surface and co-immunoprecipitated with a peripheral acrosomal membrane protein, spermadhesin AQN1. Recombinant UCHs, UCHL3, and isopeptidase T both reduced polyspermy when added to fertilization medium. UCHL1 was detected in the oocyte cortex but not on the sperm surface, and partially degraded within 6-8 hours after fertilization. Enucleated oocyte-somatic cell electrofusion caused polarized redistribution of cortical UCHL1. We conclude that sperm-acrosomal UCHs are involved in sperm-zona interactions and anti-polyspermy defense. Modulation of UCH-activity could help managing polyspermy during IVF and provide insight into male infertility.
Key words:
Fertilization •
Sperm •
hydrolase •
proteasome •
ubiquitin
