Junaid Kashir

Sperm PLCζ: From structure to Ca2+ oscillations, egg activation and therapeutic potential

Significant evidence now supports the assertion that cytosolic calcium oscillations during fertilization in mammalian eggs are mediated by a testis‐specific phospholipase C (PLC), termed PLC‐zeta (PLCζ) that is released into the egg following gamete fusion. Herein, we describe the current paradigm of PLCζ in this fundamental biological process, summarizing recent important advances in our knowledge of the biochemical and physiological properties of this enzyme. We describe the data suggesting that PLCζ has distinct features amongst PLCs enabling the hydrolysis of its substrate, phosphatidylinositol 4,5‐bisphosphate (PIP2) at low Ca2+ levels. PLCζ appears to be unique in its ability to target PIP2 that is present on intracellular vesicles. We also discuss evidence that PLCζ may be a significant factor in human fertility with potential therapeutic capacity.

Sperm-induced Ca2+ release during egg activation in mammals.

This review discusses the role that the sperm-specific phospholipase C zeta (PLCζ) is proposed to play during the fertilization of mammalian eggs. At fertilization, the sperm initiates development by causing a series of oscillations in cytosolic concentrations of calcium [Ca(2)] within the egg. PLCζ mimics the sperm at fertilization, causing the same pattern of Ca(2+) release as seen at fertilization. Introducing PLCζ into mouse eggs also mimics a number of other features of the way in which the fertilizing sperm triggers Ca(2+) oscillations. We discuss the localization of PLCζ within the egg and present a hypothesis about the localization of PLCζ within the sperm before the initiation of fertilization.

Distinctive malfunctions of calmodulin mutations associated with heart RyR2-mediated arrhythmic disease.

Calmodulin (CaM) is a cytoplasmic calcium sensor that interacts with the cardiac ryanodine receptor (RyR2), a large Ca(2+) channel complex that mediates Ca(2+) efflux from the sarcoplasmic reticulum (SR) to activate cardiac muscle contraction. Direct CaM association with RyR2 is an important physiological regulator of cardiac muscle excitation-contraction coupling and defective CaM-RyR2 protein interaction has been reported in cases of heart failure. Recent genetic studies have identified CaM missense mutations in patients with a history of severe cardiac arrhythmogenic disorders that present divergent clinical features, including catecholaminergic polymorphic ventricular tachycardia (CPVT), long QT syndrome (LQTS) and idiopathic ventricular fibrillation (IVF). Herein, we describe how two CPVT- (N54I & N98S) and three LQTS-associated (D96V, D130G & F142L) CaM mutations result in alteration of their biochemical and biophysical properties. Ca(2+)-binding studies indicate that the CPVT-associated CaM mutations, N54I & N98S, exhibit the same or a 3-fold reduced Ca(2+)-binding affinity, respectively, versus wild-type CaM, whereas the LQTS-associated CaM mutants, D96V, D130G & F142L, display more profoundly reduced Ca(2+)-binding affinity. In contrast, all five CaM mutations confer a disparate RyR2 interaction and modulation of [(3)H]ryanodine binding to RyR2, regardless of CPVT or LQTS association. Our findings suggest that the clinical presentation of CPVT or LQTS associated with these five CaM mutations may involve both altered intrinsic Ca(2+)-binding as well as defective interaction with RyR2.

Altered RyR2 regulation by the calmodulin F90L mutation associated with idiopathic ventricular fibrillation and early sudden cardiac death

Calmodulin (CaM) association with the cardiac muscle ryanodine receptor (RyR2) regulates excitation–contraction coupling. Defective CaM–RyR2 interaction is associated with heart failure. A novel CaM mutation (CaMF90L) was recently identified in a family with idiopathic ventricular fibrillation (IVF) and early onset sudden cardiac death. We report the first biochemical characterization of CaMF90L. F90L confers a deleterious effect on protein stability. Ca2+‐binding studies reveal reduced Ca2+‐binding affinity and a loss of co‐operativity. Moreover, CaMF90L displays reduced RyR2 interaction and defective modulation of [3H]ryanodine binding. Hence, dysregulation of RyR2‐mediated Ca2+ release via aberrant CaMF90L–RyR2 interaction is a potential mechanism that underlies familial IVF.

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 (PLCζ) as a diagnostic marker of sperm oocyte activation capacity and male fertility? SUMMARY ANSWER 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. WHAT IS KNOWN ALREADY 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. STUDY DESIGN, SIZE DURATION 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. PARTICIPANTS/MATERIALS, SETTING, METHODS Aldehyde- and methanol-fixed sperm were subject to immunofluorescence analysis following HCl exposure (pH = 0.1-0.5), acid Tyrodes 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 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. LIMITATIONS, REASONS FOR CAUTION 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. WIDER IMPLICATIONS OF THE FINDINGS 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. LARGE SCALE DATA Not applicable. STUDY FUNDING/COMPETING INTERESTS 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.

Mutations in PLCδ1 associated with hereditary leukonychia display divergent PIP2 hydrolytic function

Hereditary leukonychia is a rare genetic nail disorder characterized by distinctive whitening of the nail plate of all 20 nails. Hereditary leukonychia may exist as an isolated feature, or in simultaneous occurrence with other cutaneous or systemic pathologies. Associations between hereditary leukonychia and mutations in the gene encoding phospholipase C delta‐1 (PLCδ1) have previously been identified. However, the molecular mechanisms underlying PLCδ1 mutations and hereditary leukonychia remain uncharacterized. In the present study, we introduced hereditary leukonychia‐linked human PLCδ1 mutations (C209R, A574T and S740R) into equivalent residues of rat PLCδ1 (C188R, A553T and S719R), and investigated their effect on the biophysical and biochemical properties of the PLCδ1 protein. Our data suggest that these PLCδ1 mutations associated with hereditary leukonychia do not uniformly alter the enzymatic ability of this protein leading to loss/gain of function, but result in significantly divergent enzymatic properties. We demonstrate here for the first time the importance of PLC‐mediated calcium (Ca2+) signalling within the manifestation of hereditary leukonychia. PLCδ1 is almost ubiquitous in mammalian cells, which may explain why hereditary leukonychia manifests in association with other systemic pathologies relating to keratin expression.

Phospholipase C zeta and calcium oscillations at fertilisation: The evidence, applications, and further questions

Oocyte activation is a fundamental event at mammalian fertilisation, initiated by a series of characteristic calcium (Ca2+) oscillations in mammals. This characteristic pattern of Ca2+ release is induced in a species-specific manner by a sperm-specific enzyme termed phospholipase C zeta (PLCζ). Reduction or absence of functional PLCζ within sperm underlies male factor infertility in humans, due to mutational inactivation or abrogation of PLCζ protein expression. Underlying such clinical implications, a significant body of evidence has now been accumulated that has characterised the unique biochemical and biophysical properties of this enzyme, further aiding the unique clinical opportunities presented. Herein, we present and discuss evidence accrued over the past decade and a half that serves to support the identity of PLCζ as the mammalian sperm factor. Furthermore, we also discuss the potential novel avenues that have yet to be examined regarding PLCζ mechanism of action in both the oocyte, and the sperm. Finally, we discuss the advances that have been made regarding the clinical therapeutic and diagnostic applications of PLCζ in potentially treating male infertility as a result of oocyte activation deficiency (OAD), and also possibly more general cases of male subfertility.

Can Facebook pages be a mode of blended learning to supplement in-class teaching in Saudi Arabia?

The aim of this study was to examine the potential of a self-designed Facebook page on Neuroscience, to supplement in-class teaching as a mode of blended learning. Posts were split into multiple choice questions (MCQs), general interest articles, neuroscience-related external links and resources, and lecture notes and PowerPoint presentations. The study was divided into three distinct phases: before, during, and after the Neuroscience block. Student responses were evaluated via a self-developed questionnaire. Grades achieved by students undertaking the block in 2015 and 2014 were recorded, as were the grades achieved by the same cohort in concurrent blocks in the same year of study. Results showed that ~80% of students reported that use of the page enhanced their overall subject knowledge and exam preparation. Highest page activity occurred during the Neuroscience block. Peak activity occurred directly before summative assessments, with MCQ posts having the highest impact. The cohort of students with access to the Facebook page achieved better grades in the block compared with the previous cohort, despite similar average performance in other subjects. We demonstrate the utility of Facebook as a powerful tool for undergraduate education, supplementing in-class teaching, and assisting in exam preparation, potentially increasing average student performance.

The role and mechanism of action of sperm PLC-zeta in mammalian fertilisation

At mammalian fertilisation, the fundamental stimulus that triggers oocyte (egg) activation and initiation of early embryonic development is an acute rise of the intracellular-free calcium (Ca2+) concentration inside the egg cytoplasm. This essential Ca2+ increase comprises a characteristic series of repetitive Ca2+ oscillations, starting soon after sperm-egg fusion. Over the last 15 years, accumulating scientific and clinical evidence supports the notion that the physiological stimulus that precedes the cytosolic Ca2+ oscillations is a novel, testis-specific phospholipase C (PLC) isoform, known as PLC-zeta (PLCζ). Sperm PLCζ catalyses the hydrolysis of phosphatidylinositol 4,5-bisphosphate triggering cytosolic Ca2+ oscillations through the inositol 1,4,5-trisphosphate signalling pathway. PLCζ is the smallest known mammalian PLC isoform with the most elementary domain organisation. However, relative to somatic PLCs, the PLCζ isoform possesses a unique potency in stimulating Ca2+ oscillations in eggs that is attributed to its novel biochemical characteristics. In this review, we discuss the latest developments that have begun to unravel the vital role of PLCζ at mammalian fertilisation and decipher its unique mechanism of action within the fertilising egg. We also postulate the significant potential diagnostic and therapeutic capacity of PLCζ in alleviating certain types of male infertility.