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

• Published in: Molecular human reproduction 2017-01, Vol.23 (1), p.54-67
• Main Authors: Kashir, Junaid, Buntwal, Luke, Nomikos, Michail, Calver, Brian L, Stamatiadis, Panagiotis, Ashley, Peter, Vassilakopoulou, Vyronia, Sanders, David, Knaggs, Paul, Livaniou, Evangelia, Bunkheila, Adnan, Swann, Karl, Lai, F Anthony
• Format: Article
• Language: English
• Subjects: Acrosin - genetics; Acrosin - immunology; Animals; Antibodies - chemistry; Antibody Specificity; Antigen-Antibody Complex - chemistry; Biomarkers - metabolism; Carrier Proteins - genetics; Carrier Proteins - immunology; Fluorescent Antibody Technique - standards; Gene Expression; Humans; Infertility, Male - enzymology; Infertility, Male - genetics; Male; Mice; Oocytes - cytology; Oocytes - physiology; Phosphoinositide Phospholipase C - analysis; Phosphoinositide Phospholipase C - chemistry; Phosphoinositide Phospholipase C - genetics; Phosphoinositide Phospholipase C - immunology; Protein Binding; Protein Conformation; Seminal Plasma Proteins - genetics; Seminal Plasma Proteins - immunology; Sperm-Ovum Interactions - physiology; Spermatozoa - enzymology; Spermatozoa - pathology; Swine; Tissue Fixation - methods
• ISSN: 1360-9947; 1460-2407
• DOI: 10.1093/molehr/gaw073

Is it possible to improve clinical visualization of phospholipase C zeta (PLCζ) as a diagnostic marker of sperm oocyte activation capacity and male fertility? Poor PLCζ visualization efficacy using current protocols may be due to steric or conformational occlusion of native PLCζ, hindering antibody access, and is significantly enhanced using antigen unmasking/retrieval (AUM) protocols. Mammalian oocyte activation is mediated via a series of intracellular calcium (Ca ) oscillations induced by sperm-specific PLCζ. PLCζ 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 PLCζ antibody specificity and detection protocols. Two PLCζ polyclonal antibodies, with confirmed PLCζ specificity, were employed in mouse, porcine and human sperm. Experiments evaluated PLCζ 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. 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. Despite high specificity for native PLCζ following immunoblotting using epitope-specific polyclonal PLCζ 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 PLCζ 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 PLCζ 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 PLCζ visualization efficacy may be due to steric or conformational occlusion of native PLCζ, hindering antibody access. Importantly, examination of sperm from individual donors revealed that AUM differentiall