The mammalian sperm factor phospholipase C zeta is critical for early embryo division and pregnancy in humans and mice.

STUDY QUESTION: Are sperm phospholipase C zeta (PLCζ) profiles linked to the quality of embryogenesis and pregnancy? SUMMARY ANSWER: Sperm PLCζ levels in both mouse and humans correlate with measures of ideal embryogenesis whereby minimal levels seem to be required to result in successful pregnancy. WHAT IS KNOWN ALREADY: While causative factors underlying male infertility are multivariable, cases are increasingly associated with the efficacy of oocyte activation, which in mammals occurs in response to specific profiles of calcium (Ca2+) oscillations driven by sperm-specific PLCζ. Although sperm PLCζ abrogation is extensively linked with human male infertility where oocyte activation is deficient, less is clear as to whether sperm PLCζ levels or localization underlies cases of defective embryogenesis and failed pregnancy following fertility treatment. STUDY DESIGN, SIZE, DURATION: A cohort of 54 couples undergoing fertility treatment were recruited at the assisted reproductive technology laboratory at the King Faisal Hospital and Research Centre, Riyadh, Kingdom of Saudi Arabia. The recruitment criteria for males was a minimum sperm concentration of 5×106 sperm/ml, while all female patients had to have at least five oocytes. Sperm PLCζ analysis was performed in research laboratories, while semen assessments were performed, and time-lapse morphokinetic data were obtained, in the fertility clinic as part of routine treatment. The CRISPR/Cas9 system was concurrently used to induce indels and single-nucleotide mutations within the Plcζ gene to generate strains of Plcζ mutant mice. Sperm PLCζ was evaluated using immunofluorescence and immunoblotting with an antibody of confirmed consistent specificity against PLCζ. PARTICIPANTS/MATERIALS, SETTING, METHODS: We evaluated PLCζ profiles in sperm samples from 54 human couples undergoing fertility treatment in the context of time-lapse morphokinetic analysis of resultant embryos, correlating such profiles to pregnancy status. Concurrently, we generated two strains of mutant Plcζ mice using CRISPR/Cas9, and performed IVF with wild type (WT) oocytes and using WT or mutant Plcζ sperm to generate embryos. We also assessed PLCζ status in WT and mutant mice sperm in the context of time-lapse morphokinetic analysis and breeding outcomes. MAIN RESULTS AND THE ROLE OF CHANCE: A significant (P ≤ 0.05) positive relationship was observed between both PLCζ relative fluorescence and relative density with the times taken for both the second cell division (CC2) (r = 0.26 and r = 0.43, respectively) and the third cell division (S2) (r = 0.26). Examination of localization patterns also indicated significant correlations between the presence or absence of sperm PLCζ and CC2 (r = 0.27 and r = -0.27, respectively; P ≤ 0.025). Human sperm PLCζ levels were at their highest in the ideal times of CC2 (8-12 h) compared to time ranges outside the ideal timeframe (<8 and >12 h) where levels of human sperm PLCζ were lower. Following assignment of PLCζ level thresholds, quantification revealed a significantly higher (P ≤ 0.05) rate of successful pregnancy in values larger than the assigned cut-off for both relative fluorescence (19% vs 40%, respectively) and relative density (8% vs 54%, respectively). Immunoblotting indicated a single band for PLCζ at 74 kDa in sperm from WT mice, while a single band was also observed in sperm from heterozygous of Plcζ mutant mouse sperm, but at a diminished intensity. Immunofluorescent analysis indicated the previously reported (Kashir et al., 2021) fluorescence patterns in WT sperm, while sperm from Plcζ mutant mice exhibited a significantly diminished and dispersed pattern at the acrosomal region of the sperm head. Breeding experiments indicated a significantly reduced litter size of mutant Plcζ male mice compared to WT mice, while IVF-generated embryos using sperm from mutant Plcζ mice exhibited high rates of polyspermy, and resulted in significantly reduced numbers of these embryos reaching developmental milestones. LIMITATIONS, REASONS FOR CAUTION: The human population examined was relatively small, and should be expanded to examine a larger multi-centre cohort. Infertility conditions are often multivariable, and it was not possible to evaluate all these in human patients. However, our mutant Plcζ mouse experiments do suggest that PLCζ plays a significant role in early embryo development. WIDER IMPLICATIONS OF THE FINDINGS: We found that minimal levels of PLCζ within a specific range were required for optimal early embryogenesis, correlating with increased pregnancy. Levels of sperm PLCζ below specific thresholds were associated with ineffective embryogenesis and lower pregnancy rates, despite eliciting successful fertilization in both mice and humans. To our knowledge, this represents the first time that PLCζ levels in sperm have been correlated to prognostic measures of embryogenic efficacy and pregnancy rates in humans. Our data suggest for the first time that the clinical utilization of PLCζ may stand to benefit not just a specific population of male infertility where oocyte activation is completely deficient (wherein PLCζ is completely defective/abrogated), but also perhaps the larger population of couples seeking fertility treatment. STUDY FUNDING/COMPETING INTEREST(S): J.K. is supported by a faculty start up grant awarded by Khalifa University (FSU-2023-015). This study was also supported by a Healthcare Research Fellowship Award (HF-14-16) from Health and Care Research Wales (HCRW) to J.K., 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) for J.K. and A.M.A. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Genetic interaction between TTG2 and AtPLC1 reveals a role for phosphoinositide signaling in a co-regulated suite of Arabidopsis epidermal pathways.

The TTG2 transcription factor of Arabidopsis regulates a set of epidermal traits, including the differentiation of leaf trichomes, flavonoid pigment production in cells of the inner testa (or seed coat) layer and mucilage production in specialized cells of the outer testa layer. Despite the fact that TTG2 has been known for over twenty years as an important regulator of multiple developmental pathways, little has been discovered about the downstream mechanisms by which TTG2 co-regulates these epidermal features. In this study, we present evidence of phosphoinositide lipid signaling as a mechanism for the regulation of TTG2-dependent epidermal pathways. Overexpression of the AtPLC1 gene rescues the trichome and seed coat phenotypes of the ttg2-1 mutant plant. Moreover, in the case of seed coat color rescue, AtPLC1 overexpression restored expression of the TTG2 flavonoid pathway target genes, TT12 and TT13/AHA10. Consistent with these observations, a dominant AtPLC1 T-DNA insertion allele (plc1-1D) promotes trichome development in both wild-type and ttg2-3 plants. Also, AtPLC1 promoter:GUS analysis shows expression in trichomes and this expression appears dependent on TTG2. Taken together, the discovery of a genetic interaction between TTG2 and AtPLC1 suggests a role for phosphoinositide signaling in the regulation of trichome development, flavonoid pigment biosynthesis and the differentiation of mucilage-producing cells of the seed coat. This finding provides new avenues for future research at the intersection of the TTG2-dependent developmental pathways and the numerous molecular and cellular phenomena influenced by phospholipid signaling.

Molecular Mechanism of Oocyte Activation in Mammals: Past, Present, and Future Directions.

During mammalian fertilization, repetitive intracellular Ca2+ increases known as Ca2+ oscillations occur. These oscillations are considered crucial for successful fertilization and subsequent embryonic development. Numerous researchers have endeavored to elucidate the factors responsible for inducing Ca2+ oscillations across various mammalian species. Notably, sperm-specific phospholipase C zeta (PLCζ) emerged as a prominent candidate capable of initiating Ca2+ oscillations, particularly in mammals. Genetic mutation of PLCζ in humans results in the absence of Ca2+ oscillations in mouse oocytes. Recent studies further underscored PLCζ's significance, revealing that sperm from PLCζ-deficient (Plcz1-/-) mice fail to induce Ca2+ oscillations upon intracytoplasmic sperm injection (ICSI). Despite these findings, observations from in vitro fertilization (IVF) experiments using Plcz1-/- sperm revealed some residual intracellular Ca2+ increases and successful oocyte activation, hinting at potential alternative mechanisms. In this review, we introduced the current hypothesis surrounding oocyte activation in mammals, informed by contemporary literature, and probed into the enigmatic mechanisms underlying mammalian fertilization-induced oocyte activation.

Phospholipase C Zeta in Human Spermatozoa: A Systematic Review on Current Development and Clinical Application.

During fertilization, the fusion of the spermatozoa with the oocytes causes the release of calcium from the oocyte endoplasmatic reticulum. This, in turn, triggers a series of calcium ion (Ca2+) oscillations, a process known as oocyte activation. The sperm-specific factor responsible for oocyte activation is phospholipase C zeta (PLCζ). Men undergoing intracytoplasmic sperm injection (ICSI) with their spermatozoa lacking PLCζ are incapable of generating Ca2+ oscillation, leading to fertilization failure. The immunofluorescence assay is the most used technique to assess the expression and localization of PLCζ and to diagnose patients with reduced/absent ability to activate the oocytes. In these patients, the use of assisted oocyte activation (AOA) technique can help to yield successful ICSI results and shorten the time of pregnancy. However, the production of a stable PLCζ recombinant protein represents a new powerful therapeutic approach to treating individuals with this condition. We aim to conduct a systematic review focusing on the expression, level, and localization of PLCζ, discussing the novel genetic mutation associated with its impairment. In addition, we highlight the benefits of AOA, looking at new and less invasive methods to diagnose and treat cases with PLCζ dysfunction.

Phospholipase PLCE1 Promotes Transcription and Phosphorylation of MCM7 to Drive Tumor Progression in Esophageal Cancer.

Phospholipase C epsilon 1 (PLCE1) is a well-established susceptibility gene for esophageal squamous cell carcinoma (ESCC). Identification of the underlying mechanism(s) regulated by PLCE1 could lead to a better understanding of ESCC tumorigenesis. In this study, we found that PLCE1 enhances tumor progression by regulating the replicative helicase MCM7 via two pathways. PLCE1 activated PKCα-mediated phosphorylation of E2F1, which led to the transcriptional activation of MCM7 and miR-106b-5p. The increased expression of miR-106b-5p, located in intron 13 of MCM7, suppressed autophagy and apoptosis by targeting Beclin-1 and RBL2, respectively. Moreover, MCM7 cooperated with the miR-106b-25 cluster to promote PLCE1-dependent cell-cycle progression both in vivo and in vitro. In addition, PLCE1 potentiated the phosphorylation of MCM7 at six threonine residues by the atypical kinase RIOK2, which promoted MCM complex assembly, chromatin loading, and cell-cycle progression. Inhibition of PLCE1 or RIOK2 hampered MCM7-mediated DNA replication, resulting in G1-S arrest. Furthermore, MCM7 overexpression in ESCC correlated with poor patient survival. Overall, these findings provide insights into the role of PLCE1 as an oncogenic regulator, a promising prognostic biomarker, and a potential therapeutic target in ESCC. SIGNIFICANCE: PLCE1 promotes tumor progression in ESCC by activating PKCα-mediated phosphorylation of E2F1 to upregulate MCM7 and miR-106b-5p expression and by potentiating MCM7 phosphorylation by RIOK2.

Arabidopsis thaliana phosphoinositide-specific phospholipase C 2 is required for Botrytis cinerea proliferation.

Phospholipase C (PLC) plays a key role in lipid signaling during plant development and stress responses. PLC activation is one of the earliest responses during pathogen perception. Arabidopsis thaliana contains seven PLC encoding genes (AtPLC1 to AtPLC7) and two pseudogenes (AtPLC8 and AtPLC9), being AtPLC2 the most abundant isoform with constitutive expression in all plant organs. PLC has been linked to plant defense signaling, in particular to the production of reactive oxygen species (ROS). Previously, we demonstrated that AtPLC2 is involved in ROS production via the NADPH oxidase isoforms RBOHD activation during stomata plant immunity. Here we studied the role of AtPLC2 on plant resistance against the necrotrophic fungus Botrytis cinerea, a broad host-range and serious agricultural pathogen. We show that the AtPLC2-silenced (amiR PLC2) or null mutant (plc2-1) plants developed smaller B. cinerea lesions. Moreover, plc2-1 showed less ROS production and an intensified SA-dependent signaling upon infection, indicating that B. cinerea uses AtPLC2-triggered responses for a successful proliferation. Therefore, AtPLC2 is a susceptibility (S) gene that facilitates B. cinerea infection and proliferation.

Phosphatidylinositol-specific phospholipase C can decrease Müller cell viability and suppress its phagocytic activity by modulating PI3K/AKT signaling pathway.

Bacillus cereus endophthalmitis is a devastating eye infection that causes rapid blindness through the release of extracellular tissue-destructive exotoxins. The phagocytic and antibacterial functions of ocular cells are the keys to limiting ocular bacterial infections. In a previous study, we identified a new virulence gene, plcA-2 (different from the original plcA-1 gene), that was strongly associated with the plcA gene of Listeria monocytogenes. This plcA gene had been confirmed to play an important role in phagocytosis. However, how the Bc-phosphatidylinositol-specific phospholipase C (PI-PLC) proteins encoded by the plcA-1/2 genes affect phagocytes remains unclear in B. cereus endophthalmitis. Here, we found that the enzymatic activity of Bc-PI-PLC-A2 was approximately twofold higher than that of Bc-PI-PLC-A1, and both proteins inhibited the viability of Müller cells. In addition, PI-PLC proteins reduced phagocytosis of Müller cells by decreasing the phosphorylation levels of key proteins in the PI3K/AKT signaling pathway. In conclusion, we showed that PI-PLC proteins contribute to inhibit the viability of and suppress the phagocytosis of Müller cells, providing new insights into the pathogenic mechanism of B. cereus endophthalmitis.

Novel variants in ACTL7A and PLCZ1 are associated with male infertility and total fertilization failure.

Total fertilization failure (TFF), which refers to fertilization failure in all mature oocytes, accounting for 5%-10% of in vitro fertilization (IVF) cycles and 1%-3% of intracytoplasmic sperm injection (ICSI) cycles in human. In this study, we recruited three unrelated primary infertile men with repeated cycles of TFF and performed whole-exome sequencing to identify the potential pathogenic variants. We identified homozygous or compound-heterozygous variants of paternal-effect genes ACTL7A and PLCZ1 that followed a Mendelian recessive inheritance pattern. Novel homozygous nonsense variant in ACTL7A [c.C146G: p.S49*] was identified in case 1, who came from a consanguineous family. Ultrastructural observation of ACTL7A-mutated spermatozoa by transmission electron microscopy (TEM) indicated that apparent increased thickness of perinuclear matrix and the acrosome was detached from the nuclear envelop. Besides, two novel compound-heterozygous variants in PLCZ1 were identified in case 2 [c.1174+3A>C:p.?; c.A1274G:p.N425S] and case 3 [c.136-1G>C:p.?; c.G1358A:p.G453D]. Mutated spermatozoa from case 2 with reduced expression of PLCZ1 showed apparent acrosome detachment by TEM analysis. And ICSI with assisted oocyte activation (ICSI-AOA) treatment can partly rescue the TFF. Taken together, our findings revealed that novel biallelic variants in the paternal-effect genes ACTL7A and PLCZ1 were associated with human TFF, which expanding the spectrum of genetic causes and facilitating the genetic diagnosis of male infertility with TFF.

Mutations in PLCZ1 induce male infertility associated with polyspermy and fertilization failure.

PURPOSE: To investigate the genetic causes of polyspermy and total fertilization failure (TFF) in two independent male patients suffering from male infertility. METHODS: Immunofluorescence (IF) staining was used to detect the localization of the PLCζ protein in sperm and the maternal pronucleus in the zygote. Genomic DNA samples were extracted from the peripheral blood of patients and their families. The ExAC database was used to identify the frequency of corresponding mutations. The PLCZ1 mutations were validated by Sanger sequencing. The pathogenicity of the identified mutations and their possible effects on the protein were assessed using in silico tools and molecular modeling. RESULTS: We identified a reported homozygous mutation c.588C > A (p.Cys196Ter) and a compound heterozygous mutation c.2 T > C(p.Met1Thr)/c.590G > A (p.Arg197His) with one novel mutation in PLCZ1. The IF results showed that these multipronuclear zygotes formed as a result of polyspermy. In silico analysis predicted that the mutations result in disease-causing proteins. IF staining revealed that PLCζ is abnormally localized in the sperm samples from the two affected patients. Assisted oocyte activation (AOA) successfully rescued polyspermy and TFF and achieved pregnancy in two patients with the PLCZ1 mutation. CONCLUSION: We identified a homozygous mutation in PLCZ1 (c.588C > A [p.Cys196Ter]) in a male patient with polyspermy after in vitro fertilization (IVF) as well as a compound heterozygous mutation c.2 T > C(p.Met1Thr)/c.590G > A (p.Arg197His) with one novel mutation in a male patient with fertilization failure after intracytoplasmic sperm injection (ICSI), and we provide evidence that the homozygous mutation can cause polyspermy and the compound heterozygous mutation can cause fertilization failure.

Cryptic mutations of PLC family members in brain disorders: recent discoveries and a deep-learning-based approach.

Phospholipase C (PLC) is an essential isozyme involved in the phosphoinositide signalling pathway, which maintains cellular homeostasis. Gain- and loss-of-function mutations in PLC affect enzymatic activity and are therefore associated with several disorders. Alternative splicing variants of PLC can interfere with complex signalling networks associated with oncogenic transformation and other diseases, including brain disorders. Cells and tissues with various mutations in PLC contribute different phosphoinositide signalling pathways and disease progression, however, identifying cryptic mutations in PLC remains challenging. Herein, we review both the mechanisms underlying PLC regulation of the phosphoinositide signalling pathway and the genetic variation of PLC in several brain disorders. In addition, we discuss the present challenges associated with the potential of deep-learning-based analysis for the identification of PLC mutations in brain disorders.

PLCE1 as a diagnostic and prognostic biomarker by promoting the growth and progression of oral squamous cell carcinoma.

OBJECTIVE: This study aimed to explore the role of phospholipase C epsilon1 (PLCE1) in the growth and progression of oral squamous cell carcinoma (OSCC) and determine its potential as a biomarker with respect to diagnosis, prognosis and treatment of OSCC. METHODS: The expression level of PLCE1 in tissue specimens was detected by immunohistochemistry (182 OSCC cases and 76 controls) and its relationship to clinicopathological parameters was analyzed. Then, the diagnostic value of PLCE1 in OSCC was verified by constructing the receiver operating characteristic (ROC) curve. Kaplan-Meier and Cox analysis were performed to investigate the role of PLCE1 in predicting the prognosis of OSCC patients. Furthermore, the effects of PLCE1 on the occurrence and development of OSCC were revealed by knocking down the level of PLCE1. RESULTS: PLCE1 was mainly located in the cytoplasm of OSCC cells, and its level in OSCC tissues was obviously higher than in adjacent normal tissues. While the expression of PLCE1 did not correlate with clinicopathological parameters of OSCC. The area under the ROC curve (AUC) of PLCE1 was 0.865 with a sensitivity of 75.8% and a specificity of 78.8%. Besides, high expression of PLCE1 suggested a worse prognosis in OSCC patients than those with low expression. The knockdown of PLCE1 obviously inhibited proliferation, migration, and invasion of OSCC cells, and induce G0 cell cycle phase arrest and apoptosis, thus preventing the progression of OSCC. CONCLUSION: PLCE1 may cause carcinogenesis and development of OSCC, which provide a novel possibility in diagnosis, prognosis and treatment of OSCC.

SPERM FACTORS AND EGG ACTIVATION: ICSI and the discovery of the sperm factor and PLCZ1.

The discovery of PLCZ1 nearly 20 years ago as the primary Ca2+ oscillation-inducing factor in the sperm of mammals represented a significant breakthrough in our quest to elucidate the molecules and pathways that promote egg activation during fertilization. The advent of the intracytoplasmic sperm injection (ICSI) technique, which made fertilization possible without sperm capacitation, acrosome reaction, and gamete fusion, strengthened the research that led to the discovery of PLCZ1 and became an essential clinical tool for humans. The use of ICSI combined with the detection of PLCZ1 expression and mutations in infertile patients established the fundamental role of PLCZ1 in human fertility while leading to the discovery of novel components of the perinuclear theca, the site of the residence of PLCZ1 in sperm before fertilization. Remarkably, the more extensive use of ICSI in species other than humans and mice revealed poor success and exposed gaps in our understanding of PLCZ1 release and/or activation. Similarly, fertilization using sperm from mouse models lacking Plcz1 has produced striking results whose true implications are yet to be determined. Nevertheless, answers to these unresolved questions will produce a complete picture of the adaptations and molecular players that mammalian species employ to ensure the success of the triggering event of embryo development that has linked generations since the beginning of times.

SPERM FACTORS AND EGG ACTIVATION: The structure and function relationship of sperm PLCZ1.

In 2002, sperm-specific phospholipase C zeta1 (PLCZ1) was discovered and through these 20 years, it has been established as the predominant sperm oocyte-activating factor. PLCZ1 cRNA expression or direct protein microinjection into mammalian oocytes triggers calcium (Ca2+) oscillations indistinguishable from those observed at fertilization. The imperative role of PLCZ1 in oocyte activation is revealed by the vast number of human mutations throughout the PLCZ1 gene that have been identified and directly linked with certain forms of male infertility due to oocyte activation deficiency. PLCZ1 is the smallest PLC in size, comprising four N-terminal EF-hand domains, followed by X and Y catalytic domains, which are separated by the XY-linker, and ending with a C-terminal C2 domain. The EF hands are responsible for the high Ca2+ sensitivity of PLCZ1. The X and Y catalytic domains are responsible for the catalysis of the phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] substrate to produce the Ca2+-mobilising messenger, inositol 1,4,5-trisphosphate (IP3), while the XY-linker plays multiple roles in the unique mode of PLCZ1 action. Finally, the C2 domain has been proposed to facilitate the anchoring of PLCZ1 to intracellular vesicles through its direct interactions with specific phosphoinositides. This review discusses recent advances in the structure and function relationship of PLCZ1 and the potential binding partners of this important sperm-specific protein in the sperm and oocyte. The unravelling of all the remaining hidden secrets of sperm PLCZ1 should help us to understand the precise mechanism of fertilization, as well as enabling the diagnosis and treatment of currently unknown forms of PLCZ1 -linked human infertility.

SPERM FACTORS AND EGG ACTIVATION: The phenotype of PLCZ1-deficient mice.

In 2002, a report suggested that oocyte activation is induced by Plcz1 in mouse oocytes, which prompted great interest in exploring the role of sperm PLCZ1. Thus, PLCZ1 loss-of-function experiments became a crucial tool for addressing this subject. Although the only option to completely delete a target protein in fully functional spermatozoa is to use gene-deficient animals, Plcz1-deficient mice were not reported until 2017. Challenges to obtain suitable in vivo models have been related to altered expression of Capza3, a neighbor gene to Plcz1 locus in mammalian genomes that is required for spermatogenesis. With the advancement of genome-editing technologies, two groups independently and simultaneously produced Plcz1 mutant mouse lines, which were the first animal models to be artificially and reliably deficient for sperm PLCZ1. All Plcz1 mutant mouse lines display normal spermatogenesis and, surprisingly, subfertility rather than complete infertility. Moreover, analysis of oocyte Ca2+ dynamics indicates that mouse PLCζ1 is an essential sperm-derived oocyte activation factor via intracytoplasmic sperm injection, as PLCZ1 deficiency causes a complete lack of Ca2+ oscillations. This seemingly contradictory phenotype can be explained by atypical Ca2+ oscillations that are provoked slowly and less frequently in the case of fertilization accompanied by physiological sperm-egg fusion. These findings not only raise new questions concerning the sperm basic biology, by clearly demonstrating the existence of a PLCZ1-independent oocyte activation mechanism in mice, but also have implications for the treatment and phenotypic interpretation of patients presenting oocyte activation failure.

SPERM FACTORS AND EGG ACTIVATION: Phospholipase C zeta (PLCZ1) and the clinical diagnosis of oocyte activation deficiency.

Oocyte activation deficiency (OAD) remains the predominant cause of total/low fertilization rate in assisted reproductive technology. Phospholipase C zeta (PLCZ1) is the dominant sperm-specific factor responsible for triggering oocyte activation in mammals. OAD has been linked to numerous PLCZ1 abnormalities in patients experiencing failed in vitro fertilization or intracytoplasmic sperm injection cycles. While significant efforts have enhanced our understanding of the clinical relevance of PLCZ1, and the potential effects of genetic variants upon functionality, our ability to apply PLCZ1 in a diagnostic or therapeutic role remains limited. Artificial oocyte activation is the only option for patients experiencing OAD but lacks a reliable diagnostic approach. Immunofluorescence analysis has revealed that the levels and localization patterns of PLCZ1 within sperm can help us to indirectly diagnose a patient's ability to induce oocyte activation. Screening of the gene encoding PLCZ1 protein is also critical if we are to fully determine the extent to which genetic factors might play a role in the aberrant expression and/or localization patterns observed in infertile patients. Collectively, these findings highlight the clinical potential of PLCZ1, both as a prognostic indicator of OAD and eventually as a therapeutic agent. In this review, we focus on our understanding of the association between OAD and PLCZ1 by discussing the localization and expression of this key protein in human sperm, the potential genetic causes of OAD, and the diagnostic tools that are currently available to us to identify PLCZ1 deficiency and select patients that would benefit from targeted therapy.

A broad clinical spectrum of PLCε1-related kidney disease and intrafamilial variability.

BACKGROUND: The phenotypic and genotypic spectrum and kidney outcome of PLCε1-related kidney disease are not well known. We attempted to study 25 genetically confirmed cases of PLCε1-related kidney disease from 11 centers to expand the clinical spectrum and to determine the relationship between phenotypic and genotypic features, kidney outcome, and the impact of treatment on outcome. METHODS: Data regarding demographics, clinical and laboratory characteristics, histopathological and genetic test results, and treatments were evaluated retrospectively. RESULTS: Of 25 patients, 36% presented with isolated proteinuria, 28% with nephrotic syndrome, and 36% with chronic kidney disease stage 5. Twenty patients underwent kidney biopsy, 13 (65%) showed focal segmental glomerulosclerosis (FSGS), and 7 (35%) showed diffuse mesangial sclerosis (DMS). Of the mutations identified, 80% had non-missense, and 20% had missense; ten were novel. No clear genotype-phenotype correlation was observed; however, significant intrafamilial variations were observed in three families. Patients with isolated proteinuria had significantly better kidney survival than patients with nephrotic syndrome at onset (p = 0.0004). Patients with FSGS had significantly better kidney survival than patients with DMS (p = 0.007). Patients who presented with nephrotic syndrome did not respond to any immunosuppressive therapy; however, 4/9 children who presented with isolated proteinuria showed a decrease in proteinuria with steroids and/or calcineurin inhibitors. CONCLUSION: PLCε1-related kidney disease may occur in a wide clinical spectrum, and genetic variations are not associated with clinical presentation or disease course. However, clinical presentation and histopathology appear to be important determinants for prognosis. Immunosuppressive medications in addition to angiotensin-converting enzyme inhibitors may be beneficial for selected patients. "A higher resolution version of the Graphical abstract is available as Supplementary information".

SPERM FACTORS AND EGG ACTIVATION: Fertilization failure after human ICSI and the clinical potential of PLCZ1.

Two decades have passed since the discovery of phospholipase C zeta (PLCZ1) as the sperm oocyte-activating factor. At present, there is a general consensus that PLCZ1 is responsible for triggering the calcium (Ca2+) oscillations necessary to start the oocyte activation process in mammals. One proof is that abnormal, reduced, or absent PLCZ1 in human spermatozoa leads to fertilization failure (FF) after intracytoplasmic sperm injection (ICSI). ICSI is the most effective assisted reproduction technique and enables overcoming almost all male infertility conditions. Despite fertilization rates of up to 80%, FF does occur in 1-3% of ICSI cycles, which leaves these patients with few options for obtaining genetically related offspring. Assisted oocyte activation (AOA) using Ca+2 ionophores has emerged as a useful treatment option for these patients. While AOA has been proven very beneficial for the treatment of sperm-related FF, some cases of female-related FF cannot be overcome by AOA. Therefore, the development of appropriate diagnostic tests that predict the prognosis of AOA treatment would be advantageous to improve the clinical management of these patients and shorten the time to pregnancy. The aim of this review is to provide an up-to-date overview of the genetic causes of FF after ICSI and to discuss the advantages and disadvantages of using PLCZ1 as a diagnostic marker or therapeutic molecule in comparison with currently available diagnostic tests and treatments.

Plcz1 Deficiency Decreased Fertility in Male Mice Which Is Associated with Sperm Quality Decline and Abnormal Cytoskeleton in Epididymis.

Phospholipase C zeta1 (Plcz1) was known to be a physiological factor in sperm that activates oocytes to complete meiosis by triggering Ca2+ oscillations after fertilisation. However, the role of male Plcz1 in spermatogenesis and early embryo development in progeny has been controversial. Plcz1 knockout (Plcz1-/-) mouse model (Plcz1m3 and Plcz1m5) was generated by using the CRISPR-Cas9 system. The fertility of Plcz1-/- mice was evaluated by analysing the number of offsprings, sperm quality, pathological changes in the testis and epididymis. RNA-seq and RT-PCR were performed to screen differentially expressed genes and signalling pathways related to fertility in Plcz1-/- mice. Further mechanism was explored by using Plcz1-/- cells. Plcz1 knockout led to hypofertility in male mice. In particular, a significant time delay in development and polyspermy was found in eggs fertilized by both Plcz1m3 and Plcz1m5 sperm. Interestingly, a decline in sperm quality combined with pathological changes in epididymis was found in Plcz1m3 mice but not in Plcz1m5 mice. Notably, abnormal cytoskeleton appears in epididymis of Plcz1m3 mice and Plcz1-/- cells. Cytoskeleton damage of epididymis is involved in fertility decline of males upon Plcz1 deficiency in this model.

Prediction of genetic alteration of phospholipase C isozymes in brain disorders: Studies with deep learning.

Genetic mutations leading to the development of various diseases, such as cancer, diabetes, and neurodegenerative disorders, can be attributed to multiple mechanisms and exposure to diverse environments. These disorders further increase gene mutation rates and affect the activity of translated proteins, both phenomena associated with cellular responses. Therefore, maintaining the integrity of genetic and epigenetic information is critical for disease suppression and prevention. With the advent of genome sequencing technologies, large-scale genomic data-based machine learning tools, including deep learning, have been used to predict and identify somatic inactivation or negative dominant expression of target genes in various diseases. Although deep learning studies have recently been highlighted for their ability to distinguish between the genetic information of diseases, conventional wisdom is also necessary to explain the correlation between genotype and phenotype. Herein, we summarize the current understanding of phosphoinositide-specific phospholipase C isozymes (PLCs) and an overview of their associations with genetic variation, as well as their emerging roles in several diseases. We also predicted and discussed new findings of cryptic PLC splice variants by deep learning and the clinical implications of the PLC genetic variations predicted using these tools.