Sperm-Surface ATP in Boar Spermatozoa is Required for Fertilization: Relevance to Sperm Proteasomal Function

Abstract

Extracellular ATP has been implicated in a number of cellular events, including mammalian sperm function. The complement of ATP-dependent sperm proteins includes six subunits of the 26S proteasome, a multi-subunit protease specific to ubiquitinated substrate-proteins. Proteolysis of ubiquitinated proteins by the 26S proteasome is necessary for the success of mammalian fertilization, including but not limited to acrosomal exocytosis (AE) and spermzona pellucida (ZP) penetration. The 26S proteasome is uniquely present on the sperm acrosomal surface during mammalian, ascidian, and invertebrate fertilization. The proteasome is a multi-subunit protease complex of B2 MDa composed of the 19S regulatory complex and a 20S proteolytic core. Integrity of the 19S complex is maintained by six 19S ATPase subunits (PSMC1 through PSMC6). Consequently, we hypothesized that fertilization will be blocked by the depletion of sperm-surface associated ATP (ssATP). Depletion of ssATP by the Solanum tuberosum apyrase, a 49 kDa, non-cell permeant enzyme, significantly reduced the ATP content measured by an adapted luminescence-ATP assay from which all permeabilizing agents were excluded. Addition of active apyrase to porcine in vitro fertilization (IVF) medium caused a concentration dependent reduction in the overall fertilization rate. No such outcomes were observed in control groups using heat-inactivated apyrase. Apyrase treatment altered the band pattern of 19S ATPase subunits PSMC1 (Rpt2) and PSMC4 (Rpt3) in Western blotting, suggesting that it had an effect on the integrity of the sperm proteasomal 19S complex. Apyrase only altered the proteasomal core activities slightly, since these activities are not directly dependent on external ATP. In contrast, sperm treatment with MG132, a specific inhibitor of the proteasomal core chymotrypsin-like activity, inhibited the target proteolytic activity, but also induced a compensatory elevation in proteasomal peptidylglutamyl peptide hydrolase activity. Altogether, the present data provide an important missing piece of evidence in support of the ssATP-dependent, proteasomal-proteolytic model of sperm-ZP interactions.

Sperm Proteasome as a Putative Egg Coat Lysin in Mammals

“…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 ).…”

“…There is evidence that sperm proteasomes are exposed onto the sperm surface and remain associated with acrosomal membranes during sperm-ZP penetration. Boar sperm proteasomes are associated with acrosomal membranes and matrix before AE and remain associated with the inner acrosomal membrane after AE (Morales et al 2004 ;Zimmerman and Sutovsky 2009 ;Yi et al 2009 ;Yi et al 2010a , b ). Proteasomal activity is present in motile boar spermatozoa (Yi et al 2009 ).…”

“…Boar sperm proteasomes are associated with acrosomal membranes and matrix before AE and remain associated with the inner acrosomal membrane after AE (Morales et al 2004 ;Zimmerman and Sutovsky 2009 ;Yi et al 2009 ;Yi et al 2010a , b ). Proteasomal activity is present in motile boar spermatozoa (Yi et al 2009 ). Adenosine triphosphate (ATP) is essential for the integrity of the 26S proteasome, which is maintained by the six 19S ATPase subunits.…”

“…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 ).…”

“…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 ).…”