Role of the sperm proteasome during fertilization and gamete interaction in the mouse

Abstract

In this work, we have investigated the role of the sperm proteasome during in vitro fertilization (IVF) and gamete interaction in the mouse. Proteasome activity was measured in extract and intact sperm using a specific substrate. In addition, sperm were treated with specific proteasome inhibitors and evaluated during IVF, binding to the zona pellucida, and progesterone- and zona pellucida-induced acrosome reactions. In other experiments, sperm membrane proteins were obtained resuspending them in Triton X-114, shaking vigorously and let standing by 4 hr. Soluble sperm proteins were partitioned in the aqueous phase and sperm membrane proteins in the detergent phase. In both phases, proteasome activity was measured. Labeling of cell surface sperm proteins was carried out with the cell-impermeable NHS-LC biotin, extracted with Triton X-114, and mixing with avidin-agarose beads. Nonpermeabilized sperm were incubated with an anti-proteasome monoclonal antibody and evaluated by indirect immunofluorescence. The results indicate that sperm extracts as well as intact sperm had proteasome activity; the sperm proteasome was involved in IVF, specifically during sperm-zona pellucida binding and the acrosome reaction; soluble sperm membrane proteins exhibited proteasome activity; biotin experiments indicated the presence of proteasomes on the sperm surface, which was corroborated by indirect immunofluorescence experiments. All these observations indicate that the mouse sperm proteasome participates in the binding to the zona pellucida and the acrosome reaction and that there is a pool of proteasomes located on the sperm head.

Sperm Proteasome as a Putative Egg Coat Lysin in Mammals

“…In most of the mammals studied, only spermatozoa with an intact acrosome bind to the ZP, in a speciesspecifi c manner. The fertilizing spermatozoan interaction with the zona glycoprotein that serves as sperm receptor (ZP3/ZPC in mouse/human and ZPB/ZPC complex in pig) triggers the AE via signal transduction events in the sperm acrosome, such as the opening of T-type, low voltage-activated Ca 2+ channels, causing an infl ux of Ca 2+ and the activation of heterotrimeric G proteins (Pasten et al 2005 ;Ikawa et al 2010 ). An increase in intracellular pH, resulting in sustained Ca 2+ infl ux, which drives AE, causes the release or exposure of acrosomal enzymes and, when combined with vigorous sperm motility, allows the spermatozoon to penetrate the ZP and fuse with the oocyte plasma membrane, the oolemma (Pasten et al 2005 ;Ikawa et al 2010 ).…”

“…The role of the UPS has been well documented in the reproductive processes of several mammalian species. For example, multi-enzymatic sperm protease complexes with similar sedimentation coeffi cients as purifi ed proteasomes from other tissues have been isolated from mouse (Pasten et al 2005 ), pig (Zimmerman and Sutovsky 2009 ), and human (Morales et al 2003 ;Morales et al 2004 ;Baker et al 2007 ) spermatozoa. Accordingly, the substrate-specifi c enzymatic activities (trypsinlike, chymotrypsin-like, and peptidylglutamyl peptide-hydrolyzing activities) of these sperm proteasomes have been recorded in mice (Pasten et al 2005 ;Bedard et al 2011 ;Rivkin et al 2009 ), pigs (Sutovsky et al 2004 ;Yi et al 2007a ;Yi et al 2009 ), and humans (Morales et al 2003;Chakravarty et al 2008 ;Kong et al 2009 ).…”

“…The sperm-associated proteasomes are tethered to the acrosomal surface in mice (Pasten et al 2005 ), pigs (Sutovsky et al 2004 ;Yi et al 2009 ), and humans (Morales et al 2004 ). The proteasomes probably become associated with the spermatozoa inner and outer acrosomal membranes during acrosomal biogenesis at the spermatid stage (Rivkin et al 2009 ).…”

“…It appears that the extracellular proteasomes exposed on the acrosomal surface of mammalian spermatozoa are involved in the ZP-induced AE and are able to directly interact with the ZP during fertilization (Sutovsky et al 2004 ;Yi et al 2009 ). The proteasomes located on the cell surface could participate in sperm-ZP binding in some mammalian species, such as humans (Chakravarty et al 2008 ;Naz and Dhandapani 2010 ) and mice (Pasten et al 2005 ). Then, the intracellular and extracellular proteasomes would blend into the acrosomal shroud and participate during the AE.…”

“…Then, the intracellular and extracellular proteasomes would blend into the acrosomal shroud and participate during the AE. Sperm proteasomes have been shown to be essential for acrosomal function and sperm-zona penetration during fertilization in several mammalian species such as mouse, pig, and human (Pasten et al 2005 ;Kong et al 2009 ;Zimmerman et al 2011 ). It is possible that the proteasomes modulating these cellular events could participate in other steps of fertilization as well.…”