title: Antigen unmasking enhances visualization efficacy of the oocyte activation factor, phospholipase C zeta, in mammalian sperm.
abstract: 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 (Ca2+) 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 differentially affects observable PLCζ fluorescence, and the proportion of sperm exhibiting detectable PLCζ fluorescence in sperm from different males.
  
  
  
  Direct correlation of fertility outcomes with the level of PLCζ in the sperm samples studied was not available. Such analyses would be required in future to determine whether the improved methodology for PLCζ visualization we propose would indeed reflect fertility status.
  
  
  
  We propose that AUM alters conformational interactions to enhance PLCζ epitope availability and visualization efficacy, supporting prospective application of AUM to reduce misinterpretation in clinical diagnosis of PLCζ-linked male infertility. Our current results suggest that it is perhaps prudent that previous studies investigating links between PLCζ 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 PLCζ appears to be kept in an inactive form in the sperm.
  
  
  
  Not applicable.
  
  
  
  J.K. is supported by a Health Fellowship award from the National Institute for Social Care and Health Research (NISCHR). M.N. is supported by a Marie Curie Intra-European Research Fellowship award. This work was also partly funded by a research grant from Cook Medical Technologies LLC. There are no competing financial interests to declare.
authors.affiliation: College of Biomedical and Life Sciences, Schools of Bioscience & Medicine, Cardiff University, Cardiff, UK kashirj@cardiff.ac.uk lait@cardiff.ac.uk.|Alfaisal University, College of Medicine, Riyadh, Saudi Arabia.|King Faisal Specialist Hospital & Research Center, Department of Comparative Medicine, Riyadh, Saudi Arabia.
authors.forename: Junaid
authors.initials: J
authors.lastname: Kashir
authors.affiliation: College of Biomedical and Life Sciences, Schools of Bioscience & Medicine, Cardiff University, Cardiff, UK.
authors.forename: Luke
authors.initials: L
authors.lastname: Buntwal
authors.affiliation: Qatar University, College of Medicine, PO BOX 2713, Doha, Qatar.|School of Medicine, Cardiff University, UK.
authors.forename: Michail
authors.initials: M
authors.lastname: Nomikos
authors.affiliation: College of Biomedical and Life Sciences, Schools of Bioscience & Medicine, Cardiff University, Cardiff, UK.
authors.forename: Brian L
authors.initials: BL
authors.lastname: Calver
authors.affiliation: College of Biomedical and Life Sciences, Schools of Bioscience & Medicine, Cardiff University, Cardiff, UK.
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Stamatiadis
authors.affiliation: Wales Fertility Institute, University Hospital Wales, Cardiff, UK.
authors.forename: Peter
authors.initials: P
authors.lastname: Ashley
authors.affiliation: National Center for Scientific Research 'Demokritos', 15310 Aghia Paraskevi, Greece.
authors.forename: Vyronia
authors.initials: V
authors.lastname: Vassilakopoulou
authors.affiliation: Wales Fertility Institute, University Hospital Wales, Cardiff, UK.
authors.forename: David
authors.initials: D
authors.lastname: Sanders
authors.affiliation: Wales Fertility Institute, University Hospital Wales, Cardiff, UK.
authors.forename: Paul
authors.initials: P
authors.lastname: Knaggs
authors.affiliation: National Center for Scientific Research 'Demokritos', 15310 Aghia Paraskevi, Greece.
authors.forename: Evangelia
authors.initials: E
authors.lastname: Livaniou
authors.affiliation: Wales Fertility Institute, University Hospital Wales, Cardiff, UK.
authors.forename: Adnan
authors.initials: A
authors.lastname: Bunkheila
authors.affiliation: College of Biomedical and Life Sciences, Schools of Bioscience & Medicine, Cardiff University, Cardiff, UK.
authors.forename: Karl
authors.initials: K
authors.lastname: Swann
authors.affiliation: College of Biomedical and Life Sciences, Schools of Bioscience & Medicine, Cardiff University, Cardiff, UK kashirj@cardiff.ac.uk lait@cardiff.ac.uk.
authors.forename: F Anthony
authors.initials: FA
authors.lastname: Lai
mesh_terms: D000176:Acrosin / Q000235:genetics / Q000276:immunology; D000818:Animals; D000906:Antibodies / Q000737:chemistry; D000918:Antibody Specificity; D000936:Antigen-Antibody Complex / Q000737:chemistry; D015415:Biomarkers / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000276:immunology; D005455:Fluorescent Antibody Technique / Q000592:standards*; D015870:Gene Expression; D006801:Humans; D007248:Infertility, Male / Q000201:enzymology* / Q000235:genetics; D008297:Male; D051379:Mice; D009865:Oocytes / Q000166:cytology / Q000502:physiology; D054801:Phosphoinositide Phospholipase C / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D011485:Protein Binding; D011487:Protein Conformation; D029589:Seminal Plasma Proteins / Q000235:genetics / Q000276:immunology; D013084:Sperm-Ovum Interactions / Q000502:physiology*; D013094:Spermatozoa / Q000201:enzymology* / Q000473:pathology; D013552:Swine; D016707:Tissue Fixation / Q000379:methods
chemical_list: D000906:Antibodies; D000936:Antigen-Antibody Complex; D015415:Biomarkers; D002352:Carrier Proteins; D029589:Seminal Plasma Proteins; C519840:WBP2NL protein, human; C499235:PLCZ1 protein, human; D054801:Phosphoinositide Phospholipase C; D000176:Acrosin
keywords: antigen unmasking/retrieval; biomarker; diagnostic; fertilization; male infertility; oocyte activation; phospholipase C zeta; sperm