SRF2016 POSTER SESSIONS (1) (64 abstracts)
Antigen unmasking improves visualisation efficacy of phospholipase C zeta (PLCζ) in mammalian sperm to enable diagnostic applicability for evaluating PLCζ-dependent human oocyte activation deficiency
Junaid Kashir 1, , Luke Buntwal 1 , Michail Nomikos 1 , Brian Calver 1 , Panagiotis Stamatiadis 1 , Peter Ashley 4 , David Sanders 4 , Paul Knaggs 4 , Adnan Bunkheila 4 , Karl Swann 1 & Francis Anthony Lai 1
1Cardiff University, Cardiff, UK; 2Alfaisal University, Riyadh, Saudi Arabia; 3King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia; 4Wales Fertility Institute, University Hospital Wales, Cardiff, UK.
Introduction: Mammalian oocyte activation is mediated via a series of intracellular calcium oscillations induced by a sperm-specific phospholipase C, PLCzeta (PLCζ). PLCζ presents significant promise as a clinical therapeutic for some forms of male infertility. However, the utility of PLCζ as a potent diagnostic tool for human sperm remains undefined. Furthermore, considerable variation in reported PLCζ localization patterns in sperm highlight the necessity for improvement in antibody specificity and detection protocols.
Materials and methods: Two PLCζ antibodies were employed in mouse, porcine, and human sperm. Human sperm was obtained following informed, written consent with full ethical approval. Antibodies against sperm-specific proteins, PAWP and acrosin, were used as controls. Ejaculated human sperm (n=15) was subject to density gradient washing. Mouse sperm (n=3) was obtained via epididymal puncture, while porcine sperm (n=3) was supplied commercially. Aldehyde- or methanol-fixed sperm were subject to PLCζ immunofluorescent analysis (>300 cells, n=3) following either HCl exposure (pH=0.1–0.5), acid Tyrodes solution (AT) exposure (pH=2.5), or heating in 10mM sodium citrate solution (pH=6.0).
Results and discussion: Despite high specificity of antibodies to native PLCζ following immunoblotting, immunofluorescent visualization efficacy in sperm from all three species was poor, whereas post-acrosomal WW-binding protein (PAWP) and acrosin exhibited relatively impressive results. Antigen unmasking/retrieval protocols (AUM) on aldehyde-fixed sperm significantly enhanced visualization efficacy, but exerted no significant change upon PAWP or acrosin fluorescence. Furthermore, AUM enhanced PLCζ visualization efficacy in methanol-fixed sperm. This suggests that poor PLCζ visualization efficacy may be due to strong interactions of PLCζ, occluding antibody access. Finally, examination of sperm from individual donors revealed that AUM differentially affects observable PLCζ parameters in sperm from different males, suggesting a risk of potential misdiagnosis without application of AUM.
JK and MN hold a NISCHR Health Fellowship and EU-FP7 Marie-Curie Fellowship, respectively. Cardiff University holds intellectual property rights on PLCζ.
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