Involvement of multimeric protein complexes in mediating the capacitation-dependent binding of human spermatozoa to homologous zonae pellucidae

Abstract

The recognition and binding of a free-swimming spermatozoon to an ovulated oocyte is one of the most important cellular interactions in biology. While traditionally viewed as a simple lock and key mechanism, emerging evidence suggests that this event may require the concerted action of several sperm proteins. In this study we examine the hypothesis that the activity of such proteins may be coordinated by their assembly into multimeric recognition complexes on the sperm surface. Through the novel application of blue native polyacrylamide gel electrophoresis (BN-PAGE), we tender the first direct evidence that human spermatozoa do indeed express a number of high molecular weight protein complexes on their surface. Furthermore, we demonstrate that a subset of these complexes displays affinity for homologous zonae pellucidae. Proteomic analysis of two such complexes using electrospray ionization mass spectrometry identified several of the components of the multimeric 20S proteasome and chaperonin-containing TCP-1 (CCT) complexes. The latter complex was also shown to harbor at least one putative zona pellucida binding protein, ZPBP2. Consistent with a role in the mediation of sperm-zona pellucida interaction we demonstrated that antibodies directed against individual subunits of these complexes were able to inhibit sperm binding to zona-intact oocytes. Similarly, these results were able to be recapitulated using native sperm lysates, the zona affinity of which was dramatically reduced by antibody labeling of the complex receptors, or in the case of the 20S proteasome the ubiquitinated zonae ligands. Overall, the strategies employed in this study have provided novel, causal insights into the molecular mechanisms that govern sperm-egg interaction.

Sperm Proteasome as a Putative Egg Coat Lysin in Mammals

“…In agreement with the foregoing study, Aitken and colleagues found that the proteasome is a component of a multimeric ZP-binding complex found in human spermatozoa (Redgrove et al 2011 ). They propose that sperm-ZP binding requires the concerted action of several sperm proteins that form multimeric recognition complexes on the sperm surface, which is an alternate and novel view different from the traditional simple lock-and-key mechanism of one receptor, one ligand.…”

“…It is important to note that the proteasome complex was shown as three large bands when examined by blue native polyacrylamide gel electrophoresis. Aitken et al theorize that this is caused by posttranslational modifi cations of certain subunits of the complex (α1-7, β1, and β4) that displayed charge shift signatures characteristic of tyrosine phosphorylation (Redgrove et al 2011 ). This fi nding is consistent with the proteasomal subunit phosphorylation reported in the acrosome that, according to Diaz et al, modulates the fertilizing capacity of human spermatozoa by inducing AE (Diaz et al 2007 ), suggesting that proteasome complexes may be differentially activated during the individual steps of fertilization.…”

“…The formation of these complexes on the sperm surface is purported to depend upon posttesticular maturation driven by the environmental changes to which the spermatozoa are exposed in the epididymis and within the female reproductive system. They report that human spermatozoa express a number of high molecular weight protein complexes on their surface and that subsets of these complexes display affi nity for homologous ZP (Redgrove et al 2011 ). Two of these complexes were revealed to be a chaperonin-containing TCP-1 (CCT), which harbors a putative ZP-binding protein, ZPBP2, and several components of the 20S proteasome that were found previously in an analysis of the human sperm proteome (Redgrove et al 2011 ;Johnston et al 2005 ).…”

“…Two of these complexes were revealed to be a chaperonin-containing TCP-1 (CCT), which harbors a putative ZP-binding protein, ZPBP2, and several components of the 20S proteasome that were found previously in an analysis of the human sperm proteome (Redgrove et al 2011 ;Johnston et al 2005 ). The role of these protein complexes in sperm-ZP interactions was further confi rmed when antibodies against the individual components of the complex, including proteasomal subunits, inhibited sperm binding to zonaintact oocytes (Redgrove et al 2011 ). Because many sperm proteins exhibit ZP-binding affi nity, it is possible that complexes containing these ZP-binding proteins may participate in sequential or hierarchical molecular interactions or act synergistically to ensure successful sperm-ZP binding.…”

“…They report that human spermatozoa express a number of high molecular weight protein complexes on their surface and that subsets of these complexes display affi nity for homologous ZP (Redgrove et al 2011 ). Two of these complexes were revealed to be a chaperonin-containing TCP-1 (CCT), which harbors a putative ZP-binding protein, ZPBP2, and several components of the 20S proteasome that were found previously in an analysis of the human sperm proteome (Redgrove et al 2011 ;Johnston et al 2005 ). The role of these protein complexes in sperm-ZP interactions was further confi rmed when antibodies against the individual components of the complex, including proteasomal subunits, inhibited sperm binding to zonaintact oocytes (Redgrove et al 2011 ).…”