The dynamics of PKC‐induced phosphorylation triggered by Ca2+ oscillations in mouse eggs

Fertilization of mammalian eggs is characterized by a series of Ca²⁺ oscillations triggered by a phospholipase C activity. These Ca²⁺ increases and the parallel generation of diacylglycerol (DAG) stimulate protein kinase C (PKC). However, the dynamics of PKC activity have not been directly measured in living eggs. Here, we have monitored the dynamics of PKC‐induced phosphorylation in mouse eggs, alongside Ca²⁺ oscillations, using fluorescent C‐kinase activity reporter (CKAR) probes. Ca²⁺ oscillations triggered either by sperm, phospholipase C zeta (PLCζ) or Sr²⁺ all caused repetitive increases in PKC‐induced phosphorylation, as detected by CKAR in the cytoplasm or plasma membrane. The CKAR responses lasted for several minutes in both the cytoplasm and plasma membrane then returned to baseline values before subsequent Ca²⁺ transients. High frequency oscillations caused by PLCζ led to an integration of PKC‐induced phosphorylation. The conventional PKC inhibitor, Gö6976, could inhibit CKAR increases in response to thapsigargin or ionomycin, but not the repetitive responses seen at fertilization. Repetitive increases in PKCδ activity were also detected during Ca²⁺ oscillations using an isoform‐specific δCKAR. However, PKCδ may already be mostly active in unfertilized eggs, since phorbol esters were effective at stimulating δCKAR only after fertilization, and the PKCδ‐specific inhibitor, rottlerin, decreased the CKAR signals in unfertilized eggs. These data show that PKC‐induced phosphorylation outlasts each Ca²⁺ increase in mouse eggs but that signal integration only occurs at a non‐physiological, high Ca²⁺ oscillation frequency. The results also suggest that Ca²⁺‐induced DAG formation on intracellular membranes may stimulate PKC activity oscillations at fertilization. J. Cell. Physiol. 228: 110–119, 2013. © 2012 Wiley Periodicals, Inc.     

Alterations of PLCbeta1 in mouse eggs change calcium oscillatory behavior following fertilization

Inositol 1,4,5-trisphosphate generated by the action of a phospholipase C (PLC) mediates release of intracellular Ca²⁺ that is essential for sperm-induced activation of mammalian eggs. Much attention currently focuses on the role of sperm-derived PLCζ in generating changes in egg intracellular Ca²⁺ despite the fact that PLCζ constitutes a very small fraction of the total amount of PLC in a fertilized egg. Eggs express several isoforms of PLC, but a role for an egg-derived PLC in sperm-induced Ca²⁺ oscillations has not been examined. Reducing egg PLCβ1 by a transgenic RNAi approach resulted in a significant decrease in Ca²⁺ transient amplitude, but not duration or frequency, following insemination. Furthermore, overexpressing PLCβ1 by microinjecting a Plcb1 cRNA significantly perturbed the duration and frequency of Ca²⁺ transients and disrupted the characteristic shape of the first transient. These results provide the first evidence for a role of an egg-derived PLC acting in conjunction with a sperm-derived PLCζ in egg activation.     

Proteolytic processing of phospholipase Czeta and [Ca2+]i oscillations during mammalian fertilization

Phospholipase Cζ (PLCζ) is a sperm-specific PLC capable of causing repetitive intracellular Ca²⁺ ([Ca²⁺]_i) release ([Ca²⁺]_i oscillations) in mammalian eggs. Accumulating evidence suggests that PLCζ is the sperm factor responsible for inducing egg activation. Nevertheless, some sperm fractions devoid of 72-kDa PLCζ showed [Ca²⁺]_i oscillation-inducing and PLCζ-like PLC activity (Kurokawa et al., (2005) Dev. Biol. 285, 376-392). Here, we report that PLCζ remains functional after proteolytic cleavage at the X-Y linker region. We found that N-terminal (33 and 37 kDa) and C-terminal fragments (27 kDa), presumably the result of PLCζ cleavage at the X-Y linker region, were present in fresh sperm as well as in sperm extracts and remained associated as functional complexes. Protease V8 cleaved 72-kDa PLCζ into 33/37 and 27 kDa fragments, while PLC activity and [Ca²⁺]_i oscillation-inducing activity persisted until degradation of the fragments. Immunodepletion or affinity depletion of these fragments abolished PLC activity and [Ca²⁺]_i oscillation-inducing activity from sperm extracts. Lastly, co-expression of cRNAs encoding residues 1-361 and 362-647 of mouse PLCζ, mimicking cleavage at the X-Y linker region, induced [Ca²⁺]_i oscillations and embryo development in mouse eggs. Our results support the hypothesis that PLCζ is the sole mammalian sperm factor and that its linker region may have important regulatory functions during mammalian fertilization.     

The eggstraordinary story of how life begins

More than 15 years have elapsed since the identification of phospholipase C ζ1 (PLCζ) from a genomic search for mouse testis/sperm‐specific PLCs. This molecule was proposed to represent the sperm factor responsible for the initiation of calcium (Ca²⁺) oscillations required for egg activation and embryo development in mammals. Supporting evidence for this role emerged from studies documenting its expression in all mammals and other vertebrate species, the physiological Ca²⁺ rises induced by injection of its messenger RNA into mammalian and nonmammalian eggs, and the lack of expression in infertile males that fail intracytoplasmic sperm injection. In the last year, genetic animal models have added support to its role as the long sought‐after sperm factor. In this review, we highlight the findings that demonstrated the role of Ca²⁺ as the universal signal of egg activation and the experimental buildup that culminated with the identification of PLCζ as the soluble sperm factor. We also discuss the structural–functional properties that make PLCζ especially suited to evoke oscillations in eggs. Lastly, we examine unresolved aspects of the function and regulation of PLCζ and whether or not it is the only sperm factor in mammalian sperm.     

Sperm PLCζ: From structure to Ca 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.     

PLCζ causes Ca(2+) oscillations in mouse eggs by targeting intracellular and not plasma membrane PI(4,5)P(2)

Sperm-specific phospholipase C ζ (PLCζ) activates embryo development by triggering intracellular Ca(2+) oscillations in mammalian eggs indistinguishable from those at fertilization. Somatic PLC isozymes generate inositol 1,4,5-trisphophate-mediated Ca(2+) release by hydrolyzing phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) in the plasma membrane. Here we examine the subcellular source of PI(4,5)P(2) targeted by sperm PLCζ in mouse eggs. By monitoring egg plasma membrane PI(4,5)P(2) with a green fluorescent protein-tagged PH domain, we show that PLCζ effects minimal loss of PI(4,5)P(2) from the oolemma in contrast to control PLCδ1, despite the much higher potency of PLCζ in eliciting Ca(2+) oscillations. Specific depletion of this PI(4,5)P(2) pool by plasma membrane targeting of an inositol polyphosphate-5-phosphatase (Inp54p) blocked PLCδ1-mediated Ca(2+) oscillations but not those stimulated by PLCζ or sperm. Immunolocalization of PI(4,5)P(2), PLCζ, and catalytically inactive PLCζ (ciPLCζ) revealed their colocalization to distinct vesicular structures inside the egg cortex. These vesicles displayed decreased PI(4,5)P(2) after PLCζ injection. Targeted depletion of vesicular PI(4,5)P(2) by expression of ciPLCζ-fused Inp54p inhibited the Ca(2+) oscillations triggered by PLCζ or sperm but failed to affect those mediated by PLCδ1. In contrast to somatic PLCs, our data indicate that sperm PLCζ induces Ca(2+) mobilization by hydrolyzing internal PI(4,5)P(2) stores, suggesting that the mechanism of mammalian fertilization comprises a novel phosphoinositide signaling pathway.     

The role of calcium on protein secretion of the albumen gland in Helisoma duryi (Gastropoda)

Abstract. The albumen gland of the freshwater pulmonate snail Helisoma duryi produces and secretes the perivitelline fluid, which coats fertilized eggs and provides nutrients to the developing embryos. It is known that perivitelline fluid secretion is stimulated by dopamine through the activation of a dopamine D₁‐like receptor, which in turn stimulates cAMP production leading to the secretion of perivitelline fluid. This paper examines the glandular release of perivitelline fluid and provides evidence for the role of Ca²⁺ in the regulated secretion of perivitelline fluid based on protein secretion experiments and inositol 1,4,5‐trisphosphate assays. Dopamine‐stimulated protein secretion by the albumen gland is reduced in Ca²⁺‐free medium or in the presence of plasma membrane Ca²⁺ channel blockers, although the Ca²⁺ channel subtype involved is unclear. In addition, dopamine‐stimulated protein secretion does not directly involve phospholipase C‐generated signaling pathways and Ca²⁺ release from intracellular stores. Sarcoplasmic/endoplasmic reticulum Ca²⁺‐ATPase inhibitors had little effect on protein secretion when applied alone; however, they potentiated dopamine‐stimulated protein secretion. Dantrolene, an inhibitor of ryanodine receptors, 8‐(N,N‐diethylamino)‐octyl‐3,4,5‐trimethoxybenzoate hydrochloride, a nonspecific inhibitor of intracellular Ca²⁺ channels, and 2‐aminoethyldiphenylborate, an inhibitor of inositol 1,4,5‐trisphosphate receptors, did not suppress protein secretion, suggesting Ca²⁺ release from internal stores does not directly regulate protein secretion. Thus, the influx of Ca²⁺ from the extracellular space appears to be the major pathway mediating protein secretion by the albumen gland. The results are discussed with respect to the role of Ca²⁺ in controlling exocytosis of proteins from the albumen gland secretory cells.     

Inositol 1,4,5‐trisphosphate receptor 1 degradation in mouse eggs and impact on [Ca2+]i oscillations

The initiation of normal embryo development depends on the completion of all events of egg activation. In all species to date, egg activation requires an increase(s) in the intracellular concentration of calcium ([Ca²⁺]_i), which is almost entirely mediated by inositol 1,4,5‐trisphosphate receptor 1 (IP₃R1). In mammalian eggs, fertilization‐induced [Ca²⁺]_i responses exhibit a periodic pattern that are called [Ca²⁺]_i oscillations. These [Ca²⁺]_i oscillations are robust at the beginning of fertilization, which occurs at the second metaphase of meiosis, but wane as zygotes approach the pronuclear stage, time after which in the mouse oscillations cease altogether. Underlying this change in frequency are cellular and biochemical changes associated with egg activation, including degradation of IP₃R1, progression through the cell cycle, and reorganization of intracellular organelles. In this study, we investigated the system requirements for IP₃R1 degradation and examined the impact of the IP₃R1 levels on the pattern of [Ca²⁺]_i oscillations. Using microinjection of IP₃ and of its analogs and conditions that prevent the development of [Ca²⁺]_i oscillations, we show that IP₃R1 degradation requires uniform and persistently elevated levels of IP₃. We also established that progressive degradation of the IP₃R1 results in [Ca²⁺]_i oscillations with diminished periodicity while a near complete depletion of IP₃R1s precludes the initiation of [Ca²⁺]_i oscillations. These results provide insights into the mechanism involved in the generation of [Ca²⁺]_i oscillations in mouse eggs. J. Cell. Physiol. 222:238–247, 2010. © 2009 Wiley‐Liss, Inc.     

Differential Regulation of Multiple Steps in Inositol 1,4,5-Trisphosphate Signaling by Protein Kinase C Shapes Hormone-stimulated Ca2+ Oscillations

How Ca²⁺ oscillations are generated and fine-tuned to yield versatile downstream responses remains to be elucidated. In hepatocytes, G protein-coupled receptor-linked Ca²⁺ oscillations report signal strength via frequency, whereas Ca²⁺ spike amplitude and wave velocity remain constant. IP₃ uncaging also triggers oscillatory Ca²⁺ release, but, in contrast to hormones, Ca²⁺ spike amplitude, width, and wave velocity were dependent on [IP₃] and were not perturbed by phospholipase C (PLC) inhibition. These data indicate that oscillations elicited by IP₃ uncaging are driven by the biphasic regulation of the IP₃ receptor by Ca²⁺, and, unlike hormone-dependent responses, do not require PLC. Removal of extracellular Ca²⁺ did not perturb Ca²⁺ oscillations elicited by IP₃ uncaging, indicating that reloading of endoplasmic reticulum stores via plasma membrane Ca²⁺ influx does not entrain the signal. Activation and inhibition of PKC attenuated hormone-induced Ca²⁺ oscillations but had no effect on Ca²⁺ increases induced by uncaging IP₃. Importantly, PKC activation and inhibition differentially affected Ca²⁺ spike frequencies and kinetics. PKC activation amplifies negative feedback loops at the level of G protein-coupled receptor PLC activity and/or IP₃ metabolism to attenuate IP₃ levels and suppress the generation of Ca²⁺ oscillations. Inhibition of PKC relieves negative feedback regulation of IP₃ accumulation and, thereby, shifts Ca²⁺ oscillations toward sustained responses or dramatically prolonged spikes. PKC down-regulation attenuates phenylephrine-induced Ca²⁺ wave velocity, whereas responses to IP₃ uncaging are enhanced. The ability to assess Ca²⁺ responses in the absence of PLC activity indicates that IP₃ receptor modulation by PKC regulates Ca²⁺ release and wave velocity.     

Mouse strain‐dependent egg factors regulate calcium signals at fertilization

Calcium (Ca²⁺) signals triggered at fertilization initiate resumption of the cell cycle and initial steps of embryonic development. In mammals, the sperm factor phospholipase Cζ triggers the release of Ca²⁺ from the endoplasmic reticulum (ER), initiating an oscillatory pattern of Ca²⁺ transients that is modulated by egg factors including Ca²⁺ influx channels, Ca²⁺ transporters, and phosphoinositide‐regulating enzymes. Here we compared characteristics of Ca²⁺ oscillations following in vitro fertilization (IVF) and ER Ca²⁺ stores among nine common laboratory mouse strains: CF1, C57BL6, SJL, CD1, DBA, FVB, 129X1, BALBc, 129S1, and the F1 hybrid B6129SF1. Sperm from B6SJLF1/J males was used for all IVF experiments. There were significant differences among the strains with respect to duration and maximum amplitude of the first Ca²⁺ transient, frequency of oscillations, and ER Ca²⁺ stores. With male strain held constant, the differences in Ca²⁺ oscillation patterns observed result from variation in egg factors across different mouse strains. Our results support the importance of egg‐intrinsic properties in determining Ca²⁺ oscillation patterns and have important implications for the interpretation and comparison of studies on Ca²⁺ dynamics at fertilization.     

“This is where it all started” – the pivotal role of PLCζ within the sophisticated process of mammalian reproduction: a systemic review

Mammalian reproduction is one of the most complex and fascinating biological phenomenon, which aims to transfer maternal and paternal genetic material to the next generation. At the end of oogenesis and spermatogenesis, both haploid gametes contain a single set of chromosomes ready to form the zygote, the first cell of the newly developing individual. The mature oocyte and spermatozoa remain in a quiescent state, during which the oocyte is characterized by nuclear and cytoplasmic arrest, while the spermatozoa necessitates further maturation within the epididymis and female reproductive track prior to egg fertilization. Either in vivo or in vitro, the sperm initiates a series of irreversible biochemical and physiological modifications in the oocyte. The earliest detected signal after fertilization is cytosolic Ca²⁺ oscillations, a prerequisite step for embryo development. These oscillations trigger the release of the oocyte from the second meiosis arrest towards embryogenesis, also known as “oocyte activation”. Phospholipase C zeta (PLCζ) is a unique sperm-soluble protein responsible for triggering the InsP₃/Ca²⁺ pathway within the oocyte, leading to Ca²⁺ oscillations and consequently to embryo development. The specific structure of PLCζ (compared to other PLCs) enables its specialized activity via the preserved X and Y catalytic domains, as well as distinct features such as rapid onset, high sensitivity to Ca²⁺ and cession of oscillations upon zygote formation. The emerging discoveries of PLCζ have stimulated studies focusing on the possible clinical applications of this protein in male infertility evaluation and management during IVF/ICSI. Fertilization failure is attributed to lack of oocyte second meiosis resumption, suggesting that ICSI failure may be related to impaired PLCζ activity. Microinjection of recombinant human PLCζ to human oocytes after ICSI fertilization failure may trigger Ca²⁺ oscillations and achieve successful fertilization, offering new hope for couples traditionally referred to sperm donation. However, more studies are still required prior to the routine implementation of this approach in the clinic. Directions for future studies are discussed.     

Starting a new life: sperm PLC-zeta mobilizes the Ca2+ signal that induces egg activation and embryo development: an essential phospholipase C with implications for male infertility

We have discovered that a single sperm protein, phospholipase C-zeta (PLCζ), can stimulate intracellular Ca(2+) signalling in the unfertilized oocyte ('egg') culminating in the initiation of embryonic development. Upon fertilization by a spermatozoon, the earliest observed signalling event in the dormant egg is a large, transient increase in free Ca(2+) concentration. The fertilized egg responds to the intracellular Ca(2+) rise by completing meiosis. In mammalian eggs, the Ca(2+) signal is delivered as a train of long-lasting cytoplasmic Ca(2+) oscillations that begin soon after gamete fusion and persist beyond the completion of meiosis. Sperm PLCζ effects Ca(2+) release from egg intracellular stores by hydrolyzing the membrane lipid PIP(2) and consequent stimulation of the inositol 1,4,5-trisphosphate (InsP(3) ) receptor Ca(2+) -signalling pathway, leading to egg activation and early embryogenesis. Recent advances have refined our understanding of how PLCζ induces Ca(2+) oscillations in the egg and also suggest its potential dysfunction as a cause of male infertility.     

The effect of CD137–CD137 ligand interaction on phospholipase C signaling pathway in human endothelial cells

We previously reported the emerging role of CD137–CD137L interaction in inflammation and atherosclerosis. The mechanism of CD137–CD137L interaction may be related to a variety of signaling pathways. The most important signaling pathway involves the activation of phospholipase C(PLC) which induces the diacylglycerol–protein kinase C(DAG–PKC) and the inositol trisphosphate-intracellular free calcium (IP₃-[Ca²⁺]i) pathway. In the current study, we investigated whether CD137–CD137L interaction can stimulate the PLC signaling pathway in human umbilical vein endothelial cells (HUVEC). The diacylglycerol (DAG) and inositol trisphosphate (IP₃) levels in HUVEC were measured by radioenzymatic assay. The activity of protein kinase (PKC) was detected by its ability to transfer phosphate from [γ-³²P]ATP to lysine-rich histone. The [Ca²⁺]i concentrations were measured by flow cytometric analysis. The DAG level and PKC activity were increased in a concentration-dependent, biphasic manner in HUVEC induced by anti-CD137. PKC activity was mainly in the cytosol at rest, and then translocated to the membrane when stimulated by anti-CD137. Similarly, rapid IP₃ formation induced by anti-CD137 coincided with the peak of the DAG level. Moreover, anti-CD137 induced peak [Ca²⁺]i responses including the rapid transient phase and the sustained phase. However, anti-CD137L suppressed the activation of the DAG–PKC and IP₃-[Ca²⁺]i signaling pathway, which was stimulated by anti-CD137 in HUVEC. In conclusion, the data suggested that CD137–CD137L interaction induces robust activation of the PLC signaling pathway in HUVEC.     

The electrical block to polyspermy induced by an intracellular Ca2+ increase at fertilization of the clawed frogs,Xenopus laevis and Xenopus tropicalis

Polyspermy blocking, to ensure monospermic fertilization, is necessary for normal diploid development in most animals. We have demonstrated here that monospermy in the clawed frog, Xenopus tropicalis, as well as in X. laevis, is ensured by a fast, electrical block to polyspermy on the egg plasma membrane after the entry of the first sperm, which is mediated by the positive‐going fertilization potential. An intracellular Ca²⁺ concentration ([Ca²⁺]_i) at the sperm entry site was propagated as a Ca²⁺ wave over the whole egg cytoplasm. In the X. tropicalis eggs fertilized in 10% Steinberg's solution, the positive‐going fertilization potential of +27 mV was generated by opening of Ca²⁺‐activated Cl^?‐channels (CaCCs). The fertilization was completely inhibited when the egg's membrane potential was clamped at +10 mV and 0 mV in X. tropicalis and X. laevis, respectively. In X. tropicalis, a small number of eggs were fertilized at 0 mV. In the eggs whose membrane potential was clamped below ?10 mV, a large increase in inward current, the fertilization current, was recorded and allowed polyspermy to occur. A small initial step‐like current (IS current) was observed at the beginning of the increase in the fertilization current. As the IS current was elicited soon after a small increase in [Ca²⁺]_i, this is probably mediated by the opening of CaCCs. This study not only characterized the fast and electrical polyspermy in X. tropicalis, but also explained that the initial phase of [Ca²⁺]_i increase causes IS current during the early phase of egg activation of Xenopus fertilization.     

PLCγ1–PKCγ Signaling‐Mediated Hsp90α Plasma Membrane Translocation Facilitates Tumor Metastasis

The 90‐kDa heat shock protein (Hsp90α) has been identified on the surface of cancer cells, and is implicated in tumor invasion and metastasis, suggesting that it is a potentially important target for tumor therapy. However, the regulatory mechanism of Hsp90α plasma membrane translocation during tumor invasion remains poorly understood. Here, we show that Hsp90α plasma membrane expression is selectively upregulated upon epidermal growth factor (EGF) stimulation, which is a process independent of the extracellular matrix. Abrogation of EGF‐mediated activation of phospholipase (PLCγ1) by its siRNA or inhibitor prevents the accumulation of Hsp90α at cell protrusions. Inhibition of the downstream effectors of PLCγ1, including Ca²⁺ and protein kinase C (PKCγ), also blocks the membrane translocation of Hsp90α, while activation of PKCγ leads to increased levels of cell‐surface Hsp90α. Moreover, overexpression of PKCγ increases extracellular vesicle release, on which Hsp90α is present. Furthermore, activation or overexpression of PKCγ promotes tumor cell motility in vitro and tumor metastasis in vivo, whereas a specific neutralizing monoclonal antibody against Hsp90α inhibits such effects, demonstrating that PKCγ‐induced Hsp90α translocation is required for tumor metastasis. Taken together, our study provides a mechanistic basis for the role for the PLCγ1–PKCγ pathway in regulating Hsp90α plasma membrane translocation, which facilitates tumor cell motility and promotes tumor metastasis.     

Store-operated Ca2+ entry is not required for fertilization-induced Ca2+ signaling in mouse eggs

Repetitive oscillations in cytoplasmic Ca²⁺ due to periodic Ca²⁺ release from the endoplasmic reticulum (ER) drive mammalian embryo development following fertilization. Influx of extracellular Ca²⁺ to support the refilling of ER stores is required for sustained Ca²⁺ oscillations, but the mechanisms underlying this Ca²⁺ influx are controversial. Although store-operated Ca²⁺ entry (SOCE) is an appealing candidate mechanism, several groups have arrived at contradictory conclusions regarding the importance of SOCE in oocytes and eggs. To definitively address this question, Ca²⁺ influx was assessed in oocytes and eggs lacking the major components of SOCE, the ER Ca²⁺ sensor STIM proteins, and the plasma membrane Ca²⁺ channel ORAI1. We generated oocyte-specific conditional knockout (cKO) mice for Stim1 and Stim2, and also generated Stim1/2 double cKO mice. Females lacking one or both STIM proteins were fertile and their ovulated eggs displayed normal patterns of Ca²⁺ oscillations following fertilization. In addition, no impairment was observed in ER Ca²⁺ stores or Ca²⁺ influx following store depletion. Similar studies were performed on eggs from mice globally lacking ORAI1; no abnormalities were observed. Furthermore, spontaneous Ca²⁺ influx was normal in oocytes from Stim1/2 cKO and ORAI1-null mice. Finally, we tested if TRPM7-like channels could support spontaneous Ca²⁺ influx, and found that it was largely prevented by NS8593, a TRPM7-specific inhibitor. Fertilization-induced Ca²⁺ oscillations were also impaired by NS8593. Combined, these data robustly show that SOCE is not required to support appropriate Ca²⁺ signaling in mouse oocytes and eggs, and that TRPM7-like channels may contribute to Ca²⁺ influx that was previously attributed to SOCE.     

Calcium Regulation in the Protozoan Model,Paramecium tetraurelia

Early in eukaryotic evolution, the cell has evolved a considerable inventory of proteins engaged in the regulation of intracellular Ca²⁺ concentrations, not only to avoid toxic effects but beyond that to exploit the signaling capacity of Ca²⁺ by small changes in local concentration. Among protozoa, the ciliate Paramecium may now be one of the best analyzed models. Ciliary activity and exo‐/endocytosis are governed by Ca²⁺, the latter by Ca²⁺ mobilization from alveolar sacs and a superimposed store‐operated Ca²⁺‐influx. Paramecium cells possess plasma membrane‐ and endoplasmic reticulum‐resident Ca²⁺‐ATPases/pumps (PMCA, SERCA), a variety of Ca²⁺ influx channels, including mechanosensitive and voltage‐dependent channels in the plasma membrane, furthermore a plethora of Ca²⁺‐release channels (CRC) of the inositol 1,4,5‐trisphosphate and ryanodine receptor type in different compartments, notably the contractile vacuole complex and the alveolar sacs, as well as in vesicles participating in vesicular trafficking. Additional types of CRC probably also occur but they have not been identified at a molecular level as yet, as is the equivalent of synaptotagmin as a Ca²⁺ sensor for exocytosis. Among established targets and sensors of Ca²⁺ in Paramecium are calmodulin, calcineurin, as well as Ca²⁺/calmodulin‐dependent protein kinases, all with multiple functions. Thus, basic elements of Ca²⁺ signaling are available for Paramecium.     

The alteration of PLCζ protein expression in unexplained infertile and asthenoteratozoospermic patients: A potential effect on sperm fertilization ability

Failed oocyte activation has been observed in unexplained infertile (UI) and asthenoteratozoospermic (AT) men. The deficiency of phospholipase C‐zeta (PLCζ) could be a possible reason for such failures and has not been studied yet. We investigated the expression and localization of PLCζ protein in the sperms of patients with UI and AT conditions. The relationships between PLCζ‐related parameters with male age, sperm characteristics, DNA integrity, and cellular maturity were assessed. Semen samples were collected from fertile (n = 40), UI (n = 40), and AT (n = 40) men. Subsequently, semen analysis, DNA fragmentation, hyaluronic acid‐binding ability, and PLCζ level along with its distribution were evaluated using computer‐assisted sperm analyzer, sperm chromatin structure assay (SCSA), hyaluronic acid‐binding assay (HBA), western blot analysis and immunofluorescence microscopy, respectively. Unlike SCSA, the values of HBA, and PLCζ expression were significantly reduced in UI and AT patients compared to fertile men, whereas no significant differences were observed among the experimental groups in terms of PLCζ localization patterns. The regression analysis also showed that HBA is the only variable associated with PLCζ levels. Furthermore, the correlation of male age with PLCζ localization in postacrosomal, equatorial, and acrosomal+postacrosomal+equatorial (A+PA+E) patterns, as well as the relation of normal morphology, with the (A+PA+E) pattern, remained in the regression model. Our findings indicated that reduced PLCζ level along with the increased DNA fragmentation and impaired maturation may be possible etiologies of decreased fertilization in the studied subjects.     

Polyspermy block in jellyfish eggs: Collaborative controls by Ca and MAPK

Jellyfish eggs neither undergo apparent cortical reaction nor show any significant change in the membrane potential at fertilization, but nevertheless show monospermy. Utilizing the perfectly transparent eggs of the hydrozoan jellyfish Cytaeis uchidae, here we show that the polyspermy block is accomplished via a novel mechanism: a collaboration between Ca²⁺ and mitogen-activated protein kinase (MAPK). In Cytaeis, adhesion of a sperm to the animal pole surface of an egg was immediately followed by sperm–egg fusion and initiation of an intracellular Ca²⁺ rise from this site. The elevated Ca²⁺ levels lasted for several minutes following the sperm–egg fusion. The Ca²⁺ rise proved to be necessary and sufficient for a polyspermy block, as inhibiting a Ca²⁺ rise with EGTA promoted polyspermy, and conversely, triggering a Ca²⁺ rise by inositol 1,4,5-trisphosphate (IP₃) or excess K⁺ immediately abolished the egg’s capacity for sperm–egg fusion. A Ca²⁺ rise at fertilization or by artificial stimulations evoked dephosphorylation of MAPK in eggs. The eggs in which phosphorylated MAPK was maintained by injection of mRNA for MAPK kinase kinase (Mos), like intact eggs, exhibited a Ca²⁺ rise at fertilization or by IP₃ injection, and shut down the subsequent sperm–egg fusion. However, the Mos-expressing eggs became capable of accepting sperm following the arrest of Ca²⁺ rise. In contrast, addition of inhibitors of MAPK kinase (MEK) to unfertilized eggs caused MAPK dephosphorylation without elevating Ca²⁺ levels, and prevented sperm–egg fusion. Rephosphorylation of MAPK by injecting Mos mRNA after fertilization recovered sperm attraction, which is known to be another MAPK-dependent event, but did not permit subsequent sperm–egg fusion. Thus, it is possible that MAPK dephosphorylation irreversibly blocks sperm–egg fusion and reversibly suppresses sperm attraction. Collectively, our data suggest that both the fast and late mechanisms dependent on Ca²⁺ and MAPK, respectively, ensure a polyspermy block in jellyfish eggs.     

OX40–OX40 ligand interaction may activate phospholipase C signal transduction pathway in human umbilical vein endothelial cells

Studies have recently supported the emerging role of OX40/OX40L interaction in atherosclerosis. The mechanism of OX40/OX40L interaction may be related to a variety of signal pathways. The most important signal pathway involves the activation of phospholipase C (PLC) which induces diacylglycerol–protein kinase C (DAG–PKC) and the inositol trisphosphate (IP₃)–intracellular free calcium ([Ca²⁺]_i) pathway. The aim of this work was to investigate whether OX40–OX40L interaction can stimulate the PLC signal pathway in human umbilical vein endothelial cells (HUVEC). The DAG and IP₃ level in HUVEC were measured by radio-enzymatic assay. The activity of PKC was detected by its ability to transfer phosphate from [γ-³²P]ATP to lysine-rich histone. [Ca²⁺]_i concentrations were measured by flow cytometric analysis. Results showed that the DAG level was markedly increased in a concentration-dependent, biphasic manner in HUVEC induced by OX40. The early phase was rapid, peaking at 30 s. The late phase reached the maximum level at 15 min and decayed slowly. OX40 increased PKC activity in a dose-dependent manner with two peaks at 40–50 s and 12–16 min, then decreased slowly, yet maintained a high level for at least 30 min. PKC activity was mainly in cytosol at rest and translocated from cytosol to membrane when stimulated by OX40. Similarly, OX40-induced rapid IP₃ formation coincided with the peak of DAG level. Moreover, OX40 also induced peak [Ca²⁺]_i responses including the rapid transient phase and the sustained phase. Anti-OX40L antibody significantly suppressed OX40-induced DAG–PKC and IP₃–[Ca²⁺]_i signal pathway activation in HUVEC. In conclusion, the data suggested that OX40–OX40L interaction induced a robust stimulation of phospholipase C signal transduction pathway in HUVEC.