Evidence for the Participation of Sperm Proteasomes in Zona Pellucida Degradation and Zona-Induced Acrosomal Exocytosis During Fertilization in the Domestic Pig.

Abstract

Realization of importance of the ubiquitin-proteasome pathway (UPP) in mammalian fertilization is continuing to grow with every new publication. The UPP is known to participate in multiple steps of mammalian fertilization, including sperm capacitation, acrosomal exocytosis (AE), and sperm-zona pellucida (ZP) penetration. In the UPP, a chaperone protein ubiquitin binds in tandem fashion to proteins that need to be recycled, and targets them for proteolysis by a multi-subunit protease, the 26S proteasome. We have been studying the participation of the 26S proteasome in the ZP-induced AE and sperm-ZP penetration during porcine fertilization. The 26S proteasome is a ~2 MDa, complex consisting of a 19S regulatory complex (role: ubiquitinated substrate recognition, deubiquitination and unfolding) and the 20S core (role: substrate degradation). Three distinct proteolytic activities reside in the 20S core: trypsin-like, chymotrypsin-like and caspase-like. In the presence of proteasomal inhibitors, porcine sperm are unable to fertilize oocytes, which may be due either to inhibition of acrosomal exocytosis and/or degradation of the ZP matrix. To understand this, fresh, capacitated boar spermatozoa were incubated with solubilized porcine ZP proteins with or without proteasomal inhibitors MG 132 (inhibits chymotrypsin-like core activity), clasto-lactacystin β-lactone (targets trypsin-like activity) and epoxomicin (targets chymotrypsin-like activity). After two-hour incubation, soluble ZP proteins and exocytosed acrosomal ghosts were separated from sperm pellets by centrifugation. They were analyzed for evidence of ZP protein degradation by the sperm proteasomes and for the accumulation of undegraded sperm acrosomal proteins. Results show that solubilized ZP proteins readily bind to acrosomal caps of the capacitated boar spermatozoa and induce AE. Presence of proteasomal inhibitors during sperm-ZP coincubation protected the zona glycoprotein ZPB from being degraded, without affecting its deglycosylation. Additionally, release of the 26S proteasomes with the acrosomal ghosts was reduced in the presence of proteasomal inhibitors. Acrosomal ghost-associated proteins such as Sperm Adhesion Molecule 1 (SPAM1), Lactadherin/SP47, Beta-Acrosin, Zona Pellucida-Binding Protein 2 (ZPBP2/IAM38) and Proacrosin-binding Protein (SP32), were protected from proteasomal degradation and accumulated in the ghost fraction. Thus, it appears that proteasomes are released from the sperm acrosome during ZP-induced acrosomal exocytosis. However, since acrosomal ghost remains zona bound and surrounds the sperm head during early stages of sperm-ZP penetration, the resident acrosomal proteasomes could assist the spermatozoon during the formation of the fertilization slit. A second pool of proteasomes remains associated with the inner acrosomal membrane (IAM) of the exocytosed spermatozoa; it may provide the fertilizing spermatozoon with a mean to digest the zona, as it advances through the fertilization slit. Collectively these results explain why the inhibition of sperm proteasomal activity prevents porcine fertilization. This model provides tools to modulate fertilization in vitro and points to a new direction in which to contemplate non-hormonal contraceptive design. Supported by USDA-NRI grant #2007-01319 and by seed funding from the F21C Program of UM (PS). (platform)