Ca2+ influx-dependent refilling of intracellular Ca2+ stores determines the frequency of Ca2+ oscillations in fertilized mouse eggs

On mammalian fertilization, long-lasting Ca²⁺ oscillations are induced in the egg by the fusing spermatozoon. While each transient Ca²⁺ increase in Ca²⁺ concentration ([Ca²⁺]) in the cytosol is due to Ca²⁺ release from the endoplasmic reticulum (ER), Ca²⁺ influx from outside is required for Ca²⁺ oscillations to persist. In this study, we investigated how Ca²⁺ influx is interrelated to the cycle of Ca²⁺ release and uptake by the intracellular Ca²⁺ stores during Ca²⁺ oscillations in fertilized mouse eggs. In addition to monitoring cytosolic [Ca²⁺] with fura-2, the influx rate was evaluated using Mn²⁺ quenching technique, and the change in [Ca²⁺] in the ER lumen was visualized with a targeted fluorescent probe. We found that the influx was stimulated after each transient Ca²⁺ release and then diminished gradually to the basal level, and demonstrated that the ER Ca²⁺ stores once depleted by Ca²⁺ release were gradually refilled until the next Ca²⁺ transient to be initiated. Experiments altering extracellular [Ca²⁺] in the middle of Ca²⁺ oscillations revealed the dependence of both the refilling rate and the oscillation frequency on the rate of Ca²⁺ influx, indicating the crucial role of Ca²⁺ influx in determining the intervals of Ca²⁺ transients. As for the influx pathway supporting Ca²⁺ oscillations to persist, STIM1/Orai1-mediated store-operated Ca²⁺ entry (SOCE) may not significantly contribute, since neither known SOCE blockers nor the expression of protein fragments that interfere the interaction between STIM1 and Orai1 inhibited the oscillation frequency or the influx rate.     

Regulation of diacylglycerol production and protein kinase C stimulation during sperm- and PLCzeta-mediated mouse egg activation

Background information. At fertilization in mammalian eggs, the sperm induces a series of Ca²⁺ oscillations via the production of inositol 1,4,5‐trisphosphate. Increased inositol 1,4,5‐trisphosphate production appears to be triggered by a sperm‐derived PLCζ (phospholipase C‐ζ) that enters the egg after gamete fusion. The specific phosphatidylinositol 4,5‐bisphosphate hydrolytic activity of PLCζ implies that DAG (diacylglycerol) production, and hence PKC (protein kinase C) stimulation, also occurs during mammalian egg fertilization. Fertilization‐mediated increase in PKC activity has been demonstrated; however, its precise role is unclear. Results. We investigated PLCζ‐ and fertilization‐mediated generation of DAG in mouse eggs by monitoring plasma‐membrane translocation of a fluorescent DAG‐specific reporter. Consistent plasma‐membrane DAG formation at fertilization, or after injection of physiological concentrations of PLCζ, was barely detectable. However, when PLCζ is overexpressed in eggs, significant plasma‐membrane DAG production occurs in concert with a series of unexpected secondary high‐frequency Ca²⁺ oscillations. We show that these secondary Ca²⁺ oscillations can be mimicked in a variety of situations by the stimulation of PKC and that they can be prevented by PKC inhibition. The way PKC leads to secondary Ca²⁺ oscillations appears to involve Ca²⁺ influx and the loading of thapsigargin‐sensitive Ca²⁺ stores. Conclusions. Our results suggest that overproduction of DAG in PLCζ‐injected eggs can lead to PKC‐mediated Ca²⁺ influx and subsequent overloading of Ca²⁺ stores. These results suggest that DAG generation in the plasma membrane of fertilizing mouse eggs is minimized since it can perturb egg Ca²⁺ homoeostasis via excessive Ca²⁺ influx.     

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.     

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.     

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.     

A novel signalling mechanism for generating ca2+ oscillations at fertilization in mammals

At fertilization in mammals the sperm activates the egg by triggering a series of oscillations in the intracellular free Ca²⁺ concentration. The precise sequence of events that occur between sperm-egg contact and the increases in intracellular Ca²⁺ remains unknown. Here, we discuss recent evidence supporting the hypothesis that a cytosolic sperm protein enters the egg after gamete membrane fusion and triggers Ca²⁺ oscillations from within the egg cytoplasm. Biochemical studies suggest that there exists a novel sperm protein, named oscillin, that specifically comigrates with Ca²⁺ oscillation-inducing activity. Oscillin has been immunolocalised to the region of the sperm that first fuses with the egg. The concept of a specific protein that triggers Ca²⁺ oscillations may have wider physiological significance since sperm oscillin can induce Ca²⁺ oscillations in somatic cells, such as neurons and hepatocytes. Unravelling the novel signalling system involved in mammalian fertilization may help reveal some fundamental molecular mechanisms responsible for triggering cytoplasmic Ca²⁺ oscillations.     

The role of MATER in endoplasmic reticulum distribution and calcium homeostasis in mouse oocytes

Ca²⁺ oscillations are a hallmark of mammalian fertilization and play a central role in the activation of development. The calcium required for these oscillations is primarily derived from the endoplasmic reticulum (ER), which accumulates in clusters at the microvillar subcortex during oocyte maturation. The migration of the ER to the cortex during maturation is thought to play an important role in rendering the ER competent to generate the calcium transients, and the redistribution of ER is believed to be primarily mediated by microtubules and microfilaments. We have previously shown that the oocyte- and early embryo-restricted maternal effect gene Mater (Nlrp5) localizes to, and is required for, formation of the oocyte cytoplasmic lattices, a tubulin-containing structure that appears to play an important role in organelle positioning and distribution during oocyte maturation. Given these observations, we hypothesized that Mater may also be required for ER redistribution and Ca²⁺ homeostasis in oocytes. To test this hypothesis, we first investigated ER localization in metaphase-II Mater^tm/tm (hypomorph) oocytes and found ER clusters to be less abundant at the microvillar cortex when compared to wild type oocytes. To examine the potential mechanisms by which MATER mediates ER redistribution, we tested whether tubulin expression levels and localization were affected in the mutant oocytes and found that the Triton-insoluble fraction of tubulin was significantly decreased in Mater^tm/tm oocytes. To identify potential functional defects associated with these ER abnormalities, we next set out to investigate if the pattern of Ca²⁺ oscillations was altered in Mater^tm/tm oocytes after fertilization in vitro. Intriguingly, Ca²⁺ oscillations in Mater^tm/tm oocytes exhibited a significantly lower first peak amplitude and a higher frequency when compared to wild type oocytes. We then found that the Ca²⁺ oscillation defect in Mater^tm/tm oocytes was likely caused by a reduced amount of Ca²⁺ in the ER stores. Taken together, these observations support the hypothesis that MATER is required for ER distribution and Ca²⁺ homeostasis in oocytes, likely due to defects in lattice-mediated ER positioning and/or redistribution.     

Caspase-3-truncated type 1 inositol 1,4,5-trisphosphate receptor enhances intracellular Ca2+ leak and disturbs Ca2+ signalling

Background information. The IP₃R (inositol 1,4,5‐trisphosphate receptor) is a tetrameric channel that accounts for a large part of the intracellular Ca²⁺ release in virtually all cell types. We have previously demonstrated that caspase‐3‐mediated cleavage of IP₃R1 during cell death generates a C‐terminal fragment of 95 kDa comprising the complete channel domain. Expression of this truncated IP₃R increases the cellular sensitivity to apoptotic stimuli, and it was postulated to be a constitutively active channel. Results. In the present study, we demonstrate that expression of the caspase‐3‐cleaved C‐terminus of IP₃R1 increased the rate of thapsigargin‐mediated Ca²⁺ leak and decreased the rate of Ca²⁺ uptake into the ER (endoplasmic reticulum), although it was not sufficient by itself to deplete intracellular Ca²⁺ stores. We detected the truncated IP₃R1 in different cell types after a challenge with apoptotic stimuli, as well as in aged mouse oocytes. Injection of mRNA corresponding to the truncated IP₃R1 blocked sperm factor‐induced Ca²⁺ oscillations and induced an apoptotic phenotype. Conclusions. In the present study, we show that caspase‐3‐mediated truncation of IP₃R1 enhanced the Ca²⁺ leak from the ER. We suggest a model in which, in normal conditions, the increased Ca²⁺ leak is largely compensated by enhanced Ca²⁺‐uptake activity, whereas in situations where the cellular metabolism is compromised, as occurring in aging oocytes, the Ca²⁺ leak acts as a feed‐forward mechanism to divert the cell into apoptosis.     

Modelling mechanism of calcium oscillations in pancreatic acinar cells

We present a simple model for calcium oscillations in the pancreatic acinar cells. This model is based on the calcium release from two receptors, inositol trisphosphate receptors (IPR) and ryanodine receptors (RyR) through the process of calcium induced calcium release (CICR). In pancreatic acinar cells, when the Ca²⁺ concentration increases, the mitochondria uptake it very fast to restrict Ca²⁺ response in the cell. Afterwards, a much slower release of Ca²⁺ from the mitochondria serves as a calcium supply in the cytosol which causes calcium oscillations. In this paper we discuss a possible mechanism for calcium oscillations based on the interplay among the three calcium stores in the cell: the endoplasmic reticulum (ER), mitochondria and cytosol. Our model predicts that calcium shuttling between ER and mitochondria is a pacemaker role in the generation of Ca²⁺oscillations. We also consider the calcium dependent production and degradation of (1,4,5) inositol-trisphosphate (IP₃), which is a key source of intracellular calcium oscillations in pancreatic acinar cells. In this study we are able to predict the different patterns of calcium oscillations in the cell from sinusoidal to raised-baseline, high frequency and low-frequency baseline spiking.     

Bidirectional Ca2+ signaling occurs between the endoplasmic reticulum and acidic organelles

The endoplasmic reticulum (ER) and acidic organelles (endo-lysosomes) act as separate Ca²⁺ stores that release Ca²⁺ in response to the second messengers IP₃ and cADPR (ER) or NAADP (acidic organelles). Typically, trigger Ca²⁺ released from acidic organelles by NAADP subsequently recruits IP₃ or ryanodine receptors on the ER, an anterograde signal important for amplification and Ca²⁺ oscillations/waves. We therefore investigated whether the ER can signal back to acidic organelles, using organelle pH as a reporter of NAADP action. We show that Ca²⁺ released from the ER can activate the NAADP pathway in two ways: first, by stimulating Ca²⁺-dependent NAADP synthesis; second, by activating NAADP-regulated channels. Moreover, the differential effects of EGTA and BAPTA (slow and fast Ca²⁺ chelators, respectively) suggest that the acidic organelles are preferentially activated by local microdomains of high Ca²⁺ at junctions between the ER and acidic organelles. Bidirectional organelle communication may have wider implications for endo-lysosomal function as well as the generation of Ca²⁺ oscillations and waves.     

The roles of Ca2+, downstream protein kinases, and oscillatory signaling in regulating fertilization and the activation of development

Reviews in Developmental Biology have covered the pathways that generate the all-important intracellular calcium (Ca²⁺) signal at fertilization [Miyazaki, S., Shirakawa, H., Nakada, K., Honda, Y., 1993a. Essential role of the inositol 1,4,5-trisphosphate receptor/Ca²⁺ release channel in Ca²⁺ waves and Ca²⁺ oscillations at fertilization of mammalian eggs. Dev. Biol. 158, 62-78; Runft, L., Jaffe, L., Mehlmann, L., 2002. Egg activation at fertilization: where it all begins. Dev. Biol. 245, 237-254] and the different temporal responses of Ca²⁺ in many organisms [Stricker, S., 1999. Comparative biology of calcium signaling during fertilization and egg activation in animals. Dev. Biol. 211, 157-176]. Those reviews raise the importance of identifying how Ca²⁺ causes the events of egg activation (EEA) and to what extent these temporal Ca²⁺ responses encode developmental information. This review covers recent studies that have analyzed how these Ca²⁺ signals are interpreted by specific proteins, and how these proteins regulate various EEA responsible for the onset of development. Many of these proteins are protein kinases (CaMKII, PKC, MPF, MAPK, MLCK) whose activity is directly or indirectly regulated by Ca²⁺, and whose amount increases during late oocyte maturation. We cover biochemical progress in defining the signaling pathways between Ca²⁺ and the EEA, as well as discuss how oscillatory or multiple Ca²⁺ signals are likely to have specific advantages biochemically and/or developmentally. These emerging concepts are put into historical context, emphasizing that key contributions have come from many organisms. The intricate interdependence of Ca²⁺, Ca²⁺-dependent proteins, and the EEA raise many new questions for future investigations that will provide insight into the extent to which fertilization-associated signaling has long-range implications for development. In addition, answers to these questions should be beneficial to establishing parameters of egg quality for human and animal IVF, as well as improving egg activation protocols for somatic cell nuclear transfer to generate stem cells and save endangered species.     

Evaluation of developmental competence,nuclear and ooplasmic maturation of calf oocytes

In this study we evaluated nuclear and ooplasmic maturation of prepuberal calf oocytes to determine a possible cause for their low developmental competency. Calf oocytes resumed meiosis and arrested at the MII stage at rates similar to that of adult animals; however, zygotes derived from calf oocytes cleaved and developed at significantly lower rates. Ooplasmic maturation was assessed during oocyte maturation and fertilization. Transmission electron microscopy revealed that a majority of calf oocytes exhibited some delay in organelle migration and redistribution following maturation. Immunofluorescence microscopy showed that following IVF, a higher percentage of calf oocytes had abnormal chromatin and microtubule configurations than those of adult cattle. These anomalies were characterized by delayed formation of sperm aster and asynchronous pronuclear formation. Microfluorometry was used to characterize the Ca²⁺ responses of calf oocytes to the addition of agonists or after IVF. The addition of thimerosal demonstrated the presence of Ca²⁺ stores in calf oocytes. Injection of near threshold concentrations of inositol 1,4,5-trisphosphate (InsP₃), used to test the sensitivity of the InsP₃R, released significantly less Ca²⁺ in calf than in cow oocytes, whereas higher concentrations of InsP₃ (500 μM) released maximal [Ca²⁺]i in both oocytes. These results suggested that the Ca²⁺ content of intracellular stores was similar, but the sensitivity of the InsP₃R may be different. Following insemination, calf oocytes exhibiting [Ca²⁺]i oscillations displayed comparable amplitude and intervals to cow oocytes; however, a significantly higher number of fertilized calf oocytes failed to show oscillations. Our findings suggest that the low developmental competence of calf oocytes can be attributed, at least in part, to incomplete or delayed ooplasmic maturation. © 1996 Wiley-Liss, Inc.     

Effects of piperine and deoxyschizandrin on synchronized Ca2+ oscillations in cultured hippocampal neuronal cells

It has been reported that piperine (PIP) and deoxyschizandrin (DS) can modulate synchronized Ca²⁺ oscillations in cultured hippocampal neuronal networks. We investigated the modulation effects of four different combinations of piperine and deoxyschizandrin on synchronized Ca²⁺ oscillations in cultured hippocampal neuronal networks. The results showed that all four combinations (PIP:DS 4.9:1.9, 2.45:2.85, 7.35:0.95, and 2.45:0.95 mg/L) inhibit Ca²⁺ oscillation intensity to a similar extent. However, the first three combinations had strong inhibitory effects on the frequency of Ca²⁺ oscillations whereas the last combination (2.45:0.95 mg/L) only slightly enhanced the frequency of Ca²⁺ oscillations. We propose an improved Chay’s model to explain the mechanism of the effects of piperine and deoxyschizandrin on synchronized Ca²⁺ oscillations in cultured hippocampal neuronal cells. We concluded that deoxyschizandrin modulated synchronized Ca²⁺ oscillations in cultured hippocampal neuronal networks bidirectionally and the effect depended on concentration. Deoxyschizandrin reduced voltage-gated sodium channel conductance and ATP-sensitive potassium channel conductance, and affected the rate of exchange of intracellular calcium and the pump activity of Ca²⁺-ATPase in the endoplasmic reticulum (ER). Piperine reduced the activity of calcium release in the ER, and reduced the pump activity of calcium in the cytomembrane or enhanced the pump activity of Ca²⁺-ATPase in the ER.     

Mitochondrial regulation of [Ca2+]i oscillations during cell cycle resumption of the second meiosis of oocyte

Oocyte is arrested at metaphase of the second meiosis until fertilization switching on [Ca²⁺]i oscillations. Oocyte activation inefficiency is the most challenging problem for failed fertilization and embryonic development. Mitochondrial function and intracellular [Ca²⁺]i oscillations are two critical factors for the oocyte’s developmental potential. We aimed to understand the possible correlation between mitochondrial function and [Ca²⁺]i oscillations in oocytes. To this end, mitochondrial uncoupler CCCP which damages mitochondrial function and two small molecule mitochondrial agonists, L-carnitine (LC) and BGP-15, were used to examine the regulation of [Ca²⁺]i by mitochondrial functions. With increasing CCCP concentrations, [Ca²⁺]i oscillations were gradually diminished and high concentrations of CCCP led to oocyte death. LC enhanced mitochondrial membrane potential and [Ca²⁺]i oscillations and even improved the damage induced by CCCP, however, BGP-15 had no beneficial effect on oocyte activation. We have found that mitochondrial function plays a vital role in the generation of [Ca²⁺]i oscillations in oocytes, and thus mitochondria may interact with the ER to generate [Ca²⁺]i oscillations during oocyte activation. Improvement of mitochondrial functions with small molecules can be expected to improve oocyte activation and embryonic development in infertile patients without invasive micromanipulation.     

Crosstalk between cellular morphology and calcium oscillation patterns Insights from a stochastic computer model

Agonist-induced oscillations in the concentration of intracellular free calcium ([Ca²⁺]₁) display a wide variety of temporal and spatial patterns. In non-excitable cells, typical oscillatory patterns are somewhat cell-type specific and range from frequency-encoded, repetitive Ca²⁺ spikes to oscillations that are more sinusoidal in shape. Although the response of a cell population, even to the same stimulus, is often extremely heterogeneous, the response of the same cell to successive exposures can be remarkably similar. We propose that such ‘Ca ²⁺ fingerprints’ can be a consequence of cell-specific morphological properties. The hypothesis is tested by means of a stochastic computer simulation of a two-dimensional model for oscillatory Ca ²⁺ waves which encompasses the basic elements of the two-pool oscillator introduced by Goldbeter et al. (Goldbeter A., Dupont G., Berridge M.J. Minimal model for signal-induced Ca²⁺-oscillations and for their frequency encoding through protein phosphorylation. Proc Natl Acad Sci USA 1990; 87: 1461–1465). In the framework of our extended spatiotemporal model, single cells can display various oscillation patterns which depend on the agonist dose, Ca²⁺ diffusibility, and several morphological parameters. These are, for example, size and shape of the cell and the cell nucleus, the amount and distribution of Ca²⁺ stores, and the subcellular location of the inositol(1,4,5)-trisphosphate-generating apparatus.     

“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.     

Oscillations of cytosolic free calcium in bombesin-stimulated HIT-T15 cells

The mechanism underlying the generation of cytosolic free Ca²⁺ ([Ca²⁺_i) oscillations by bombesin, a receptor agonist activating phospholipase C, in insulin secreting HIT-T15 cells was investigated. At 25 μM, 61% of cells displayed [Ca²⁺]_i oscillations with variable patterns. The bombesin-induced [Ca²⁺]_i oscillations could last more than 1 h and glucose was required for maintaining these [Ca²⁺ fluctuations. Bombesin-evoked [Ca²⁺]_i oscillations were dependent on extracellular Ca²⁺ entry and were attenuated by membrane hype rpolarization or by L-type Ca²⁺ channel blockers. These [Ca²⁺]_i oscillations were apparently not associated with fluctuations in plasma membrane Ca²⁺ permeability as monitored by the Mn²⁺ quenching technique. 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) and 4-chloro-m-cresol, which interfere with intracellular Ca²⁺ stores, respectively, by inhibiting Ca²⁺-ATPase of endoplasmic reticulum and by affecting Ca²⁺-induced Ca²⁺ release, disrupted bombesin-induced [Ca²⁺]_i oscillations. 4-chloro-m-resol raised [Ca²⁺]_i by mobilizing an intracellular Ca²⁺ pool, an effect not altered by ryanodine. Caffeine exerted complex actions on [Ca²⁺]_i It raised [Ca²⁺]_i by promoting Ca²⁺ entry while inhibiting bombesin-elicited [Ca²⁺]_i oscillations. Our results suggest that in bombesin-elicited [Ca²⁺]_i oscillations in HIT-T15 cells: (i) the oscillations originate primarily from intracellular Ca²⁺ stores; and (ii) the Ca²⁺ influx required for maintaining the oscillations is in part membrane potential-sensitive and not coordinated with [Ca²⁺]_i oscillations. The interplay between intracellular Ca²⁺ stores and voltage-sensitive and voltage-insensitive extracellular Ca²⁺ entry determines the [Ca²⁺]_i oscillations evoked by bombesin.     

Departure gate of acidic Ca2+ confirmed

More potent, but less known than IP₃ that liberates Ca²⁺ from the ER, NAADP releases Ca²⁺ from acidic stores. The notion that TPC channels mediate this Ca²⁺ release was questioned recently by studies suggesting that TPCs are rather PI(3,5)P₂‐activated Na⁺ channels. Ruas et al (2015) now partially reconcile these views by showing that TPCs significantly conduct both cations and confirm their activation by both NAADP and PI(3,5)P₂. They attribute the failure of others to observe TPC‐dependent NAADP‐induced Ca²⁺ release in vivo to inadequate mouse models that retain partial TPC function.     

De-regulated expression of the plant glutamate receptor homolog AtGLR3.1 impairs long-term Ca2+-programmed stomatal closure

Cytosolic Ca²⁺ ([Ca²⁺]_cyt) mediates diverse cellular responses in both animal and plant cells in response to various stimuli. Calcium oscillation amplitude and frequency control gene expression. In stomatal guard cells, [Ca²⁺]_cyt has been shown to regulate stomatal movements, and a defined window of Ca²⁺ oscillation kinetic parameters encodes necessary information for long‐term stomatal movements. However, it remains unknown how the encrypted information in the cytosolic Ca²⁺ signature is decoded to maintain stomatal closure. Here we report that the Arabidopsis glutamate receptor homolog AtGLR3.1 is preferentially expressed in guard cells compared to mesophyll cells. Furthermore, over‐expression of AtGLR3.1 using a viral promoter resulted in impaired external Ca²⁺‐induced stomatal closure. Cytosolic Ca²⁺ activation of S‐type anion channels, which play a central role in Ca²⁺‐reactive stomatal closure, was normal in the AtGLR3.1 over‐expressing plants. Interestingly, AtGLR3.1 over‐expression did not affect Ca²⁺‐induced Ca²⁺ oscillation kinetics, but resulted in a failure to maintain long‐term ‘Ca²⁺‐programmed’ stomatal closure when Ca²⁺ oscillations containing information for maintaining stomatal closure were imposed. By contrast, prompt short‐term Ca²⁺‐reactive closure was not affected in AtGLR3.1 over‐expressing plants. In wild‐type plants, the translational inhibitor cyclohexamide partially inhibited Ca²⁺‐programmed stomatal closure induced by experimentally imposed Ca²⁺ oscillations without affecting short‐term Ca²⁺‐reactive closure, mimicking the guard cell behavior of the AtGLR3.1 over‐expressing plants. Our results suggest that over‐expression of AtGLR3.1 impairs Ca²⁺ oscillation‐regulated stomatal movements, and that de novo protein synthesis contributes to the maintenance of long‐term Ca²⁺‐programmed stomatal closure.     

Differential regulation of intracellular calcium oscillations by mitochondria and gap junctions

Fluctuations of intracellular Ca²⁺ ([Ca²⁺]_i) regulate a variety of cellular functions. The classical Ca²⁺ transport pathways in the endoplasmic reticulum (ER) and plasma membrane are essential to [Ca²⁺]_i oscillations. Although mitochondria have recently been shown to absorb and release Ca²⁺ during G protein-coupled receptor (GPCR) activation, the role of mitochondria in [Ca²⁺]_i oscillations remains to be elucidated. Using fluo-3-loaded human teratocarcinoma NT2 cells, we investigated the regulation of [Ca²⁺]_i oscillations by mitochondria. Both the muscarinic GPCR agonist carbachol and the ER Ca²⁺-adenosine triphosphate inhibitor thapsigargin (Tg) induced [Ca²⁺]_i oscillations in NT2 cells. The [Ca²⁺]_i oscillations induced by carbachol were unsynchronized among individual NT2 cells; in contrast, Tg-induced oscillations were synchronized. Inhibition of mitochondrial functions with either mitochondrial blockers or depletion of mitochondrial DNA eliminated carbachol—but not Tg-induced [Ca²⁺]_i oscillations. Furthermore, carbachol-induced [Ca²⁺]_i oscillations were partially restored to mitochondrial DNA-depleted NT2 cells by introduction of exogenous mitochondria. Treatment of NT2 cells with gap junction blockers prevented Tg-induced but not carbachol-induced [Ca²⁺]_i oscillations. These data suggest that the distinct patterns of [Ca²⁺]_i oscillations induced by GPCR and Tg are differentially modulated by mitochondria and gap junctions.