Molecular and enzymatic characterization of mammalian phospholipase C zeta (PLCϚ)

In mammalian oocytes, the fertilising sperm evokes intracellular calcium (Ca2+) oscillations that are essential for the initiation of egg activation and embryonic development. Although the exact mechanism leading to the initiation of Ca2+ oscillations is still unclear, accumulating evidence suggests that sperm-specific phospholipase C zeta (PLC zeta) is delivered from the fertilising sperm into the ooplasm, triggering the Ca2+ oscillations through the inositol 1,4,5-trisphosphate (InsP3) pathway. PLC zeta is the smallest known mammalian PLC isoform comprising of two EF hand, a C2 and the X and Y catalytic core domains. In this study we examined the biochemical properties of recombinant bacterially expressed mouse PLC zeta (m PLC zeta) using the well-characterised rat PLC zeta1 (rPLC zeta1) as control. Using a PtdInsP2 hydrolysis assay we showed that both isoforms had a similar Km for PtdIns(4,5)P2 and that PLC zeta had a much higher Ca2+ sensitivity, which would predict it to be active at resting Ca2+ concentrations in eggs. PLC zeta bound with high affinity to PtdIns(3,5)P2 and PtdIns(4,5)P2 even though it lacks a PH domain from its sequence, which targets PLC zeta1 to PtdIns(4,5)P2. A series of domain deletion constructs of PLC zeta were used to demonstrate the role of the EF hands on the Ca2+ sensitivity of PLC zeta and the role of C2 domain and XY linker on its binding to PtdInsP2. Luminescent PLC constructs were generated to examine their potential to elicit Ca2+ oscillations, quantifying their expression levels in mouse eggs. Anti-human PLC zeta (h PLC zeta) monoclonal antibodies were produced and their ability to block the in vitro hydrolysing activity of recombinant h PLC zeta was tested

Bonded particles models of rock plates with circular cavities in uniaxial and biaxial compression

In this paper, a distinct elements code is used to perform a numerical investigation for the size and stress gradient effects on the fracture initiation and propagation around single or pairs of pre-existing cavities in brittle rock. To investigate the rock fracture around cavities and to assess the potential of the numerical model to simulate this behavior, published laboratory physical model on granite is simulated numerically with a Bonded Particles Model (BPM). The numerical model is presented and the calibration of the BPM micro-parameters is described. Then, the calibrated BPMs are used to investigate the effect of the size of the cavity on the primary, secondary and side wall fracturing, as well as on the fracturing modes. Moreover, BPMs with two circular cavities were used to study the interaction of these holes of the same diameter and to investigate the importance of their relative distance. Finally, the simulated material was studied by biaxial tests on BPMs with a pre-existing hole

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

AbstractSignificant 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

Editorial: The role of calcium signaling in gametogenesis and early embryogenesis

Calcium homeostasis exerts a profound role in determining the quality of gametogenesis, the resultant efficacy of fertilisation and ultimately the competency of embryogenesis. Perhaps no other single ion plays such a profound and determinative role in reproductive success as calcium, since it is critical for germinal vesicle breakdown, sperm capacitation and hyperactivation, oocyte activation, maintenance of zygote calcium homeostasis and embryogenic developmental gradients. This Research Topic aimed to collect, collate, and summarize the most recent and cutting-edge advances and propositions studying the molecular determinants and effectors of calcium signalling and homeostasis throughout the early events of fertilisation and preimplantation embryogenesis, with a specific view to examine how calcium can affect the efficacy of such important phenomena.JK was supported by a Healthcare Research Fellowship Award (HF-14–16) made by Health and Care Research Wales (HCRW), alongside a National Science, Technology, and Innovation plan (NSTIP) project grant (15-MED4186-20) awarded by the King Abdulaziz City for Science and Technology (KACST)

Novel regulation of PLCζ activity via its XY-linker

The XY-linker region of somatic cell PLC (phospholipase)-β, -γ, -δ and -ϵ isoforms confers potent catalytic inhibition, suggesting a common auto-regulatory role. Surprisingly, the sperm PLCζ XY-linker does not mediate auto-inhibition. Unlike for somatic PLCs, the absence of the PLCζ XY-linker significantly diminishes both in vitro PIP2 (phosphatidylinositol 4,5-bisphosphate) hydrolysis and in vivo Ca2+-oscillation-inducing activity, revealing evidence for a novel PLCζ enzymatic mechanism

Essential Role of Sperm-Specific PLC-Zeta in Egg Activation and Male Factor Infertility: An Update.

Sperm-specific phospholipase C zeta (PLCζ) is widely considered to be the physiological stimulus responsible for generating calcium (Ca) oscillations that induce egg activation and early embryonic development during mammalian fertilization. In the mammalian testis, PLCζ expression is detected at spermiogenesis following elongated spermatid differentiation. Sperm-delivered PLCζ induces Ca release via the inositol 1,4,5-trisphosphate (InsP) signaling pathway. PLCζ is the smallest known mammalian PLC isoform identified to date, with the simplest domain organization. However, the distinctive biochemical properties of PLCζ compared with other PLC isoforms contribute to its unique potency in stimulating cytosolic Ca oscillations within mammalian eggs. Moreover, studies describing PLCζ "knockout" mouse phenotypes confirm the supreme importance of PLCζ at egg activation and monospermic fertilization in mice. Importantly, a number of clinical reports have highlighted the crucial importance of PLCζ in human fertilization by associating PLCζ deficiencies with certain forms of male factor infertility. Herein, we give an update on recent advances that have refined our understanding of how sperm PLCζ triggers Ca oscillations and egg activation in mammals, while also discussing the nature of a potential "alternative" sperm factor. We summarise PLCζ localization in mammalian sperm, and the direct links observed between defective PLCζ protein in sperm and documented cases of male infertility. Finally, we postulate how this sperm protein can be used as a potential diagnostic marker, and also as a powerful therapeutic agent for treatment of certain types of male infertility due to egg activation failure or even in more general cases of male subfertility.Qatar University student grant QUST-1-CMED-2020-3. Healthcare Research Fellowship Award (HF-14-16) made by Health and Care Research Wales (HCRW). National Science, Technology, and Innovation plan (NSTIP) project grant (15-MED4186-20) awarded by the King Abdulaziz City for Science and Technology (KACST)

Ca2+ dynamics in oocytes from naturally-aged mice

The ability of human metaphase-II arrested eggs to activate following fertilisation declines with advancing maternal age. Egg activation is triggered by repetitive increases in intracellular Ca2+ concentration ([Ca2+]i) in the ooplasm as a result of sperm-egg fusion. We therefore hypothesised that eggs from older females feature a reduced ability to mount appropriate Ca2+ responses at fertilisation. To test this hypothesis we performed the first examination of Ca2+ dynamics in eggs from young and naturally-aged mice. Strikingly, we find that Ca2+ stores and resting [Ca2+]i are unchanged with age. Although eggs from aged mice feature a reduced ability to replenish intracellular Ca2+ stores following depletion, this difference had no effect on the duration, number, or amplitude of Ca2+ oscillations following intracytoplasmic sperm injection or expression of phospholipase C zeta. In contrast, we describe a substantial reduction in the frequency and duration of oscillations in aged eggs upon parthenogenetic activation with SrCl2. We conclude that the ability to mount and respond to an appropriate Ca2+ signal at fertilisation is largely unchanged by advancing maternal age, but subtle changes in Ca2+ handling occur that may have more substantial impacts upon commonly used means of parthenogenetic activation

Construct validity of an instrument for assessment of reflective writing-based portfolios of medical students

Purpose: Assessment of reflective writing for medical students is challenging, and there is lack of an available instrument with good psychometric properties. The authors developed a new instrument for assessment of reflective writing-based portfolios and examined the construct validity of this instrument. Methods: After an extensive literature review and pilot testing of the instrument, two raters assessed the reflective writing-based portfolios from years 2 and 3 medical students (n=135) on three occasions. The instrument consists of three criteria: organization, description of an experience and reflection on the experience. We calculated the reliability of scores using generalizability theory with a fully crossed design and two facets (raters and occasions). In addition, we measured criterion validity by testing correlations with students’ scores using other assessment methods. Results: The dependability (Φ) coefficient of the portfolio scores was 0.75 using two raters on three occasions. Students’ portfolio scores represented 46.6% of the total variance across all score comparisons. The variance due to occasions was negligible, while the student– occasion interaction was small. The variance due to student–rater interaction represented 17.7%, and the remaining 27.7% of the variance was due to unexplained sources of error. The decision (D) study suggested that an acceptable dependability (Φ = 0.70 and 0.72) can be achieved by using two raters for one and two occasions, respectively. Finally, we found moderate to large effect-size correlations between students’ scores in reflective writing-based portfolios and communication skills (r = 0.47) and PBL tutorials (r = 0.50). Conclusion: We demonstrated the presence of different sources of evidence that support construct validity of the study instrument. Further studies are warranted before utilizing this instrument for summative assessment of students’ reflective writing-based portfolios in other medical schools

The Relationship between Changes in MYBPC3 Single-Nucleotide Polymorphism-Associated Metabolites and Elite Athletes’ Adaptive Cardiac Function

Athletic performance is a multifactorial trait influenced by a complex interaction of environmental and genetic factors. Over the last decades, understanding and improving elite athletes’ endurance and performance has become a real challenge for scientists. Significant tools include but are not limited to the development of molecular methods for talent identification, personalized exercise training, dietary requirements, prevention of exercise-related diseases, as well as the recognition of the structure and function of the genome in elite athletes. Investigating the genetic markers and phenotypes has become critical for elite endurance surveillance. The identification of genetic variants contributing to a predisposition for excellence in certain types of athletic activities has been difficult despite the relatively high genetic inheritance of athlete status. Metabolomics can potentially represent a useful approach for gaining a thorough understanding of various physiological states and for clarifying disorders caused by strength–endurance physical exercise. Based on a previous GWAS study, this manuscript aims to discuss the association of specific single-nucleotide polymorphisms (SNPs) located in the MYBPC3 gene encoding for cardiac MyBP-C protein with endurance athlete status. MYBPC3 is linked to elite athlete heart remodeling during or after exercise, but it could also be linked to the phenotype of cardiac hypertrophy (HCM). To make the distinction between both phenotypes, specific metabolites that are influenced by variants in the MYBPC3 gene are analyzed in relation to elite athletic performance and HCM. These include theophylline, ursodeoxycholate, quinate, and decanoyl-carnitine. According to the analysis of effect size, theophylline, quinate, and decanoyl carnitine increase with endurance while decreasing with cardiovascular disease, whereas ursodeoxycholate increases with cardiovascular disease. In conclusion, and based on our metabolomics data, the specific effects on athletic performance for each MYBPC3 SNP-associated metabolite are discussed.M.N. was supported by a QU Internal Grant “QUCG-CMED-22/23-514”. Open access publication of this article is funded by the College of Medicine, QU Health, Qatar University