Antigen unmasking enhances visualization efficacy of the oocyte activation factor, phospholipase C zeta, in mammalian sperm

STUDY QUESTION: Is it possible to improve clinical visualization of phospholipase C zeta (PLC.) as a diagnostic marker of sperm oocyte activation capacity and male fertility? SUMMARY ANSWER: Poor PLC zeta visualization efficacy using current protocols may be due to steric or conformational occlusion of native PLC zeta, hindering antibody access, and is significantly enhanced using antigen unmasking/retrieval (AUM) protocols. WHAT IS KNOWN ALREADY: Mammalian oocyte activation is mediated via a series of intracellular calcium (Ca2+) oscillations induced by sperm-specific PLC zeta. PLC zeta represents not only a potential clinical therapeutic in cases of oocyte activation deficiency but also a diagnostic marker of sperm fertility. However, there are significant concerns surrounding PLC zeta antibody specificity and detection protocols. STUDY DESIGN, SIZE DURATION: Two PLC zeta polyclonal antibodies, with confirmed PLC zeta specificity, were employed in mouse, porcine and human sperm. Experiments evaluated PLC zeta visualization efficacy, and whether AUM improved this. Antibodies against two sperm-specific proteins [post-acrosomal WW-binding protein (PAWP) and acrosin] were used as controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Aldehyde-and methanol-fixed sperm were subject to immunofluorescence analysis following HCl exposure (pH = 0.1-0.5), acid Tyrode's solution exposure (pH = 2.5) or heating in 10 mM sodium citrate solution (pH = 6.0). Fluorescence intensity of at least 300 cells was recorded for each treatment, with three independent repeats. MAIN RESULTS AND THE ROLE OF CHANCE: Despite high specificity for native PLC zeta following immunoblotting using epitope-specific polyclonal PLC zeta antibodies in mouse, porcine and human sperm, immunofluorescent visualization efficacy was poor. In contrast, sperm markers PAWP and acrosin exhibited relatively impressive results. All methods of AUM on aldehyde-fixed sperm enhanced visualization efficacy for PLC zeta compared to visualization efficacy before AUM (P < 0.05 for all AUM interventions), but exerted no significant change upon PAWP or acrosin immunofluorescence following AUM. All methods of AUM enhanced PLC zeta visualization efficacy in mouse and human methanol-fixed sperm compared to without AUM (P < 0.05 for all AUM interventions), while no significant change was observed in methanol-fixed porcine sperm before and after. In the absence of aldehyde-induced cross-linkages, such results suggest that poor PLC zeta visualization efficacy may be due to steric or conformational occlusion of native PLC zeta, hindering antibody access. Importantly, examination of sperm from individual donors revealed that AUM differentially affects observable PLC zeta fluorescence, and the proportion of sperm exhibiting detectable PLddC zeta fluorescence in sperm from different males. LIMITATIONS, REASONS FOR CAUTION: Direct correlation of fertility outcomes with the level of PLC zeta in the sperm samples studied was not available. Such analyses would be required in future to determine whether the improved methodology for PLC zeta visualization we propose would indeed reflect fertility status. WIDER IMPLICATIONS OF THE FINDINGS: We propose that AUM alters conformational interactions to enhance PLC zeta epitope availability and visualization efficacy, supporting prospective application of AUM to reduce misinterpretation in clinical diagnosis of PLC zeta-linked male infertility. Our current results suggest that it is perhaps prudent that previous studies investigating links between PLC zeta and fertility parameters are re-examined in the context of AUM, and may pave the way for future work to answer significant questions such as how PLC zeta appears to be kept in an inactive form in the sperm. LARGE SCALE DATA: Not applicable.