The equatorial subsegment in mammalian spermatozoa is enriched in tyrosine phosphorylated proteins.

The equatorial subsegment (EqSS) was originally identified by atomic force microscopy as a discrete region within the equatorial segment of Artiodactyl spermatozoa. In this investigation, we show that the EqSS is enriched in tyrosine phosphorylated proteins and present preliminary evidence for its presence in mouse and rat spermatozoa. The anti-phosphotyrosine monoclonal antibody (McAb) 4G10 bound strongly and discretely to the EqSS of permeabilized boar, ram, and bull spermatozoa. It also bound to a small patch on the posterior acrosomal region of permeabilized mouse and rat spermatozoa, suggesting that the EqSS is not restricted to the order Artiodactyla. An anti-HSPA1A (formerly Hsp70) antibody recognized the EqSS in boar spermatozoa. Immunogold labeling with McAb 4G10 localized the tyrosine phosphorylated proteins to the outer acrosomal membrane. This was verified by freeze-fracture electron microscopy, which identified the EqSS in three overlying membranes, the plasma membrane, outer acrosomal membrane, and inner acrosomal membrane. In all five species, tyrosine phosphorylated proteins became restricted to the EqSS during sperm maturation in the epididymis. The major tyrosine phosphorylated proteins in the EqSS of boar and ram spermatozoa were identified by mass spectrometry as orthologs of human SPACA1 (formerly SAMP32). Immunofluorescence with a specific polyclonal antibody localized SPACA1 to the equatorial segment in boar spermatozoa. We speculate that the EqSS is an organizing center for assembly of multimolecular complexes that initiate fusion competence in this area of the plasma membrane following the acrosome reaction.

Binding of sperm proacrosin/beta-acrosin to zona pellucida glycoproteins is sulfate and stereodependent. Synthesis of a novel fertilization inhibitor.

Specific binding of spermatozoa to the zona pellucida that surrounds mammalian eggs is a key step in the fertilization process. However, the sperm proteins that recognise zona pellucida receptors remain contentious despite longstanding research efforts to identify them. Here we present evidence that proacrosin, a tissue-specific protein found within the acrosomal vesicle of all mammalian spermatozoa, is a multifunctional protein that mediates binding of acrosome-reacted spermatozoa to zona glycoproteins via a stereospecific polysulfate recognition mechanism. Using sulfated versus non-sulfated forms of chemically defined compounds in binding assays employing native proteins in their normal cellular location or conjugated to FluoSpheres, we have attempted to identify the sulfation "code" required for recognition. Results show that protein conformation is important for specificity and that at least 2 sulfate groups are required to cross-link spatially separated docking sites on proacrosin. The consistently most effective inhibitory compounds were suramin and quercetin-3beta-d-glucoside sulfate. The results support our hypothesis that proacrosin is one of several proteins in the acrosomal matrix that retain acrosome reacted spermatozoa on the zona surface prior to penetration. They also establish, as a proof-of-principle, the feasibility of synthesising sulfated compounds of high specificity as antifertility agents for human or animal use.

Cholesterol efflux alters lipid raft stability and distribution during capacitation of boar spermatozoa.

A reduction in plasma membrane cholesterol is one of the early events that either triggers or is closely associated with capacitation of mammalian spermatozoa. In this investigation, we have examined the effects of cholesterol efflux on tyrosine phosphorylation, lipid diffusion, and raft organization in boar spermatozoa. Results show that a low level of cholesterol efflux, mediated by 5 mM methyl-beta-cyclodextrin (MBCD), enhances capacitation and induces phosphorylation of two proteins at 26 and 15 kDa without affecting sperm viability. Lipid diffusion rates under these conditions are largely unaffected except when cholesterol efflux is excessive. Low-density Triton X100-insoluble complexes (lipid rafts) were isolated from spermatozoa and found to have a restricted profile of proteins. Capacitation-associated cholesterol efflux has no effect on raft composition, but cholesterol depletion destabilizes them completely and phosphorylation is suppressed. During MBCD-mediated capacitation, the distribution of GM1 gangliosides on spermatozoa changes in a sequential manner from overlying the sperm tail to clustering on the sperm head. It is concluded that there is a safe window for removal of plasma membrane cholesterol from spermatozoa within which protein phosphorylation and polarized migration of lipid rafts take place. A preferential loss of cholesterol from the nonraft pool may be the stimulus that promotes raft clustering over the anterior sperm head.