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Phospholipase C in mouse oocytes: characterization of beta and gamma isoforms and their possible involvement in sperm-induced Ca2+ spiking.

ABSTRACT: This study involved an investigation of the role of phospholipase C (PLC) in generating repetitive Ca2+ spikes at fertilization. Using a PCR-based strategy we have demonstrated that mouse oocytes have mRNA coding for PLC beta 1, PLC beta 3 and PLC gamma isoenzymes. Furthermore, immunodetection of PLC gamma 1 using monoclonal antibodies reveals that PLC gamma 1 protein is present in mature mouse oocytes, ruling out the possibility that mRNA was being transcribed but not expressed. We were unsuccessful at detecting the presence of PLC beta protein, but the presence of this isoform can be inferred from functional studies. The PLC inhibitor, U73122, exerted an inhibitory effect on oocytes activated by spermatozoa or acetylcholine at concentrations of 10 and 30 microM respectively, while its inactive analogue had no effect. The soluble tyrosine kinase inhibitors, genistein (100 microM), herbimycin (10 microM) and geldanamycin (0.6 microM) which could affect signalling through PLC gamma hindered but never completely inhibited Ca2+ spiking in response to fertilization. We conclude that the activation of PLC to generate InsP3 may play a critical role in fertilization.

SUBMITTER: Dupont G

PROVIDER: S-EPMC1217388 | biostudies-other | 1996 Jun

REPOSITORIES: biostudies-other

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Sperm-specific post-acrosomal WW-domain binding protein (PAWP) does not cause Ca2+ release in mouse oocytes.

Project description:Mature mammalian oocytes undergo a prolonged series of cytoplasmic calcium (Ca(2+)) oscillations at fertilization that are the cause of oocyte activation. The Ca(2+) oscillations in mammalian oocytes are driven via inositol 1,4,5-trisphosphate (IP3) generation. Microinjection of the sperm-derived phospholipase C-zeta (PLCζ), which generates IP3, causes the same pattern of Ca(2+) oscillations as observed at mammalian fertilization and it is thought to be the physiological agent that triggers oocyte activation. However, another sperm-specific protein, 'post-acrosomal WW-domain binding protein' (PAWP), has also been reported to elicit activation when injected into mammalian oocytes, and to produce a Ca(2+) increase in frog oocytes. Here we have investigated whether PAWP can induce fertilization-like Ca(2+) oscillations in mouse oocytes. Recombinant mouse PAWP protein was found to be unable to hydrolyse phosphatidylinositol 4,5-bisphosphate in vitro and did not cause any detectable Ca(2+) release when microinjected into mouse oocytes. Microinjection with cRNA encoding either the untagged PAWP, or yellow fluorescent protein (YFP)-PAWP, or luciferase-PAWP fusion proteins all failed to trigger Ca(2+) increases in mouse oocytes. The lack of response in mouse oocytes was despite PAWP being robustly expressed at similar or higher concentrations than PLCζ, which successfully initiated Ca(2+) oscillations in every parallel control experiment. These data suggest that sperm-derived PAWP is not involved in triggering Ca(2+) oscillations at fertilization in mammalian oocytes.

| S-EPMC4172172 | biostudies-literature

Different Ca2+-releasing abilities of sperm extracts compared with tissue extracts and phospholipase C isoforms in sea urchin egg homogenate and mouse eggs.

Project description:A soluble phospholipase C (PLC) from boar sperm generates InsP(3) and hence causes Ca(2+) release when added to sea urchin egg homogenate. This PLC activity is associated with the ability of sperm extracts to cause Ca(2+) oscillations in mammalian eggs following fractionation. A sperm PLC may, therefore, be responsible for causing the observed Ca(2+) oscillations at fertilization. In the present study we have further characterized this boar sperm PLC activity using sea urchin egg homogenate. Consistent with a sperm PLC acting on egg PtdIns(4,5)P(2), the ability of sperm extracts to release Ca(2+) was blocked by preincubation with the PLC inhibitor U73122 or by the addition of neomycin to the homogenate. The Ca(2+)-releasing activity was also detectable in sperm from other species and in whole testis extracts. However, activity was not observed in extracts from other tissues. Moreover recombinant PLCbeta1, -gamma1, -gamma2, -delta1, all of which had higher specific activities than boar sperm extracts, were not able to release Ca(2+) in the sea urchin egg homogenate. In addition these PLCs were not able to cause Ca(2+) oscillations following microinjection into mouse eggs. These results imply that the sperm PLC possesses distinct properties that allow it to hydrolyse PtdIns(4,5)P(2) in eggs.

| S-EPMC1220908 | biostudies-other

Membrane Cholesterol Inhibits Progesterone-Mediated Sperm Function through the Possible Involvement of ABHD2.

Project description:Abhydrolase domain containing 2-acylglycerol lipase (ABHD2) was recently claimed as the membrane receptor of progesterone (P4) in sperm cells, mediating cell processes such as sperm chemotaxis and acrosome reaction. Here, we investigated the role of membrane cholesterol (Chol) on ABHD2-mediated human sperm chemotaxis. Human sperm cells were obtained from twelve normozoospemic healthy donors. ABHD2-Chol interaction was modelled by computational molecular-modelling (MM). Sperm membrane Chol content was depleted by incubating cells with cyclodextrin (CD) or augmented by the incubation with the complex between CD and Chol (CD:Chol). Cell Chol levels were quantified by liquid chromatography-mass spectrometry. Sperm migration upon P4 gradient was evaluated through the accumulation assay in a specific migration device. Motility parameters were evaluated by sperm class analyzer, whilst intracellular calcium concentration, acrosome reaction and mitochondrial membrane potential were evaluated with calcium orange, FITC-conjugated anti-CD46 antibody and JC-1 fluorescent probes, respectively. MM analysis showed the possible stable binding Chol to ABHD2, resulting in to major impact on the protein backbone flexibility. The treatment with CD was associated with a dose-dependent increase in sperm migration in a 160 nM P4 gradient, together with increase in sperm motility parameters and levels of acrosome reaction. The treatment with CD:Chol was associated with essentially opposite effects. Chol was, thus, suggested to inhibit P4-mediated sperm function through the possible inhibition of ABHD2.

| S-EPMC10253330 | biostudies-literature

Functional Characterization of Novel Bony Fish Lipoxygenase Isoforms and Their Possible Involvement in Inflammation

Project description:Eicosanoids and related compounds are pleiotropic lipid mediators, which are biosynthesized in mammals via three distinct metabolic pathways (cyclooxygenase pathway, lipoxygenase pathway, epoxygenase pathway). These mediators have been implicated in the pathogenesis of inflammatory diseases and drugs interfering with eicosanoid signaling are currently available as antiphlogistics. Eicosanoid biosynthesis has well been explored in mammals including men, but much less detailed information is currently available on eicosanoid biosynthesis in other vertebrates including bony fish. There are a few reports in the literature describing the expression of arachidonic acid lipoxygenases (ALOX isoforms) in several bony fish species but except for two zebrafish ALOX-isoforms (zfALOX1 and zfALOX2) bony fish eicosanoid biosynthesizing enzymes have not been characterized. To fill this gap and to explore the possible roles of ALOX15 orthologs in bony fish inflammation we cloned and expressed putative ALOX15 orthologs from three different bony fish species (N. furzeri, P. nyererei, S. formosus) as recombinant N-terminal his-tag fusion proteins and characterized the corresponding enzymes with respect to their catalytic properties (temperature-dependence, activation energy, pH-dependence, substrate affinity and substrate specificity with different polyenoic fatty acids). Furthermore, we identified the chemical structure of the dominant oxygenation products formed by the recombinant enzymes from different free fatty acids and from more complex lipid substrates. Taken together, our data indicate that functional ALOX isoforms occur in bony fish but that their catalytic properties are different from those of mammalian enzymes. The possible roles of these ALOX-isoforms in bony fish inflammation are discussed.

| S-EPMC9787790 | biostudies-literature

Using ZINC08918027 inhibitor to determine Aurora kinase-chromosomal passenger complex isoforms in mouse oocytes.

Project description:ObjectiveMiscarriages affect 10% of women aged 25-29, and 53% of women over 45. The primary cause of miscarriage is aneuploidy that originated in eggs. The Aurora kinase family has three members that regulate chromosome segregation. Therefore, distinguishing the roles of these isoforms is important to understand aneuploidy etiology. In meiosis, Aurora kinase A (AURKA) localizes to spindle poles, where it binds TPX2. Aurora kinase C (AURKC) localizes on chromosomes, where it replaces AURKB as the primary AURK in the chromosomal passenger complex (CPC) via INCENP binding. Although AURKA compensates for CPC function in oocytes lacking AURKB/C, it is unknown whether AURKA binds INCENP in wild type mouse oocytes. ZINC08918027 (ZC) is an inhibitor that prevents the interaction between AURKB and INCENP in mitotic cells. We hypothesized that ZC would block CPC function of any AURK isoform.ResultsZC treatment caused defects in meiotic progression and spindle building. By Western blotting and immunofluorescence, we observed that activated AURKA and AURKC levels in ZC-treated oocytes decreased compared to controls. These results suggest there is a population of AURKA-CPC in mouse oocytes. These data together suggest that INCENP-dependent AURKA and AURKC activities are needed for spindle bipolarity and meiotic progression.

| S-EPMC8900367 | biostudies-literature

Serotonin enhances excitability and gamma frequency temporal integration in mouse prefrontal fast-spiking interneurons.

Project description:The medial prefrontal cortex plays a key role in higher order cognitive functions like decision making and social cognition. These complex behaviors emerge from the coordinated firing of prefrontal neurons. Fast-spiking interneurons (FSIs) control the timing of excitatory neuron firing via somatic inhibition and generate gamma (30-100 Hz) oscillations. Therefore, factors that regulate how FSIs respond to gamma-frequency input could affect both prefrontal circuit activity and behavior. Here, we show that serotonin (5HT), which is known to regulate gamma power, acts via 5HT2A receptors to suppress an inward-rectifying potassium conductance in FSIs. This leads to depolarization, increased input resistance, enhanced spiking, and slowed decay of excitatory post-synaptic potentials (EPSPs). Notably, we found that slowed EPSP decay preferentially enhanced temporal summation and firing elicited by gamma frequency inputs. These findings show how changes in passive membrane properties can affect not only neuronal excitability but also the temporal filtering of synaptic inputs.

| S-EPMC5746342 | biostudies-literature

Ca2+ channels couple spiking to mitochondrial metabolism in substantia nigra dopaminergic neurons

Project description:How do neurons match generation of adenosine triphosphate (ATP) by mitochondria to the bioenergetic demands of regenerative activity? Although the subject of speculation, this coupling is still poorly understood, particularly in neurons that are tonically active. To help fill this gap, pacemaking substantia nigra dopaminergic neurons were studied using a combination of optical, electrophysiological and molecular approaches. In these neurons, spike-activated calcium (Ca2+) entry through Cav1 channels triggered Ca2+ release from the endoplasmic reticulum, which stimulated mitochondrial OXPHOS through two complementary Ca2+-dependent mechanisms: one mediated by the mitochondrial uniporter and another by the malate-aspartate shuttle. Disrupting either mechanism impaired the ability of dopaminergic neurons to sustain spike activity. While this feedforward control helps dopaminergic neurons meet the bioenergetic demands associated with sustained spiking, it also is responsible for their elevated oxidant stress and possibly to their decline with aging and disease.

2022-07-31 | GSE206366 | GEO

Ca2+ signalling mechanisms of the beta 2 integrin on neutrophils: involvement of phospholipase C gamma 2 and Ins(1,4,5)P3.

Project description:Engagement of beta 2 integrins triggers a tyrosine kinase-dependent intracellular mobilization and influx of Ca2+ in human neutrophils. However, the transduction pathway involved in generating this Ca2+ signal is obscure. In the present study we identified phospholipase C gamma 2 (PLC gamma 2) as one of the major proteins that was phosphorylated on tyrosine in response to beta 2 integrin activation. This beta 2 integrin-induced phosphorylation of PLC gamma 2 occurred in parallel with an increased accumulation of Ins(1,4,5)P3. The relevance of these observations for the beta 2 integrin-induced Ca2+ signal was investigated using an inhibitor of PLC signalling pathways, 1-(6-{[17 beta-3-methoxyoestra-1,3.5(10)-trien-17-yl] amino}hexyl)-1H-pyrrole-2,5-dione(U73122). U73122 dose-dependently (IC50, approx. 0.15 microM) inhibited both the beta 2 integrin-induced release of Ca2+ from intracellular stores and the subsequent influx of Ca2+ across the plasma membrane. These effects were not observed with the inactive analogue 1-(6-{[17 beta-3-methoxyoestra-1,3,5(10)-trien-17-yl] amino}hexyl)-pyrrolidine-2,5-dione (U73343). To gain further support for an involvement of PLC-induced Ins(1,4,5)P3 formation in the beta 2 integrin-induced Ca2+ signal, we searched for the molecular event(s) underlying the effects of U73122. Our experiments revealed that U73122 had no effect on either beta 2 integrin-induced tyrosine phosphorylation of PLC gamma 2 (or any of the other proteins) or on the formation of Ins(1,4,5)P3, but it reduced the Ins(1,4,5)P3-induced release of 45Ca2+ from intracellular stores of electropermeabilized cells. Taken together, the present data suggest that the beta 2 integrin-induced Ca2+ signal in human neutrophils is generated through activation of a PLC gamma 2-dependent pathway.

| S-EPMC1217502 | biostudies-other

Interaction of Phospholipase A/Acyltransferase-3 with Pex19p: A POSSIBLE INVOLVEMENT IN THE DOWN-REGULATION OF PEROXISOMES.

Project description:Phospholipase A/acyltransferase (PLA/AT)-3 (also known as H-rev107 or AdPLA) was originally isolated as a tumor suppressor and was later shown to have phospholipase A1/A2 activity. We have also found that the overexpression of PLA/AT-3 in mammalian cells results in specific disappearance of peroxisomes. However, its molecular mechanism remained unclear. In the present study, we first established a HEK293 cell line, which stably expresses a fluorescent peroxisome marker protein (DsRed2-Peroxi) and expresses PLA/AT-3 in a tetracycline-dependent manner. The treatment with tetracycline, as expected, caused disappearance of peroxisomes within 24 h, as revealed by diffuse signals of DsRed2-Peroxi and a remarkable decrease in a peroxisomal membrane protein, PMP70. A time-dependent decrease in ether-type lipid levels was also seen. Because the activation of LC3, a marker of autophagy, was not observed, the involvement of autophagy was unlikely. Among various peroxins responsible for peroxisome biogenesis, Pex19p functions as a chaperone protein for the transportation of peroxisomal membrane proteins. Immunoprecipitation analysis showed that PLA/AT-3 binds to Pex19p through its N-terminal proline-rich and C-terminal hydrophobic domains. The protein level and enzyme activity of PLA/AT-3 were increased by its coexpression with Pex19p. Moreover, PLA/AT-3 inhibited the binding of Pex19 to peroxisomal membrane proteins, such as Pex3p and Pex11?p. A catalytically inactive point mutant of PLA/AT-3 could bind to Pex19p but did not inhibit the chaperone activity of Pex19p. Altogether, these results suggest a novel regulatory mechanism for peroxisome biogenesis through the interaction between Pex19p and PLA/AT-3.

| S-EPMC4498086 | biostudies-literature

Persistent Gamma Spiking in SI Nonsensory Fast Spiking Cells Predicts Perceptual Success.

Project description:Gamma oscillations (30-55 Hz) are hypothesized to temporally coordinate sensory encoding, enabling perception. However, fast spiking interneurons (FS), key gamma generators, can be highly sensory responsive, as is the gamma band local field potential (LFP). How can FS-mediated gamma act as an impartial temporal reference for sensory encoding, when the sensory drive itself presumably perturbs the pre-established rhythm? Combining tetrode recording in SI barrel cortex with controlled psychophysics, we found a unique FS subtype that was not sensory responsive and spiked regularly at gamma range intervals (gamma regular nonsensory FS [grnsFS]). Successful detection was predicted by a further increase in gamma regular spiking of grnsFS, persisting from before to after sensory onset. In contrast, broadband LFP power, including gamma, negatively predicted detection and did not cohere with gamma band spiking by grnsFS. These results suggest that a distinct FS subtype mediates perceptually relevant oscillations, independent of the LFP and sensory drive.

| S-EPMC6763387 | biostudies-literature

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