These signals are crucial for the success of development and, in the case of mammals, health of the offspring. The mechanisms associated with fertilization that trigger these signals and the molecules that regulate their characteristic patterns vary widely. With few exceptions, a major contributor to fertilization-induced elevation in cytoplasmic Ca2+ is release from endoplasmic reticulum stores through the IP3 receptor. In some cases, Ca2+ influx from the extracellular space and/or release from alternative intracellular stores contribute to the rise in cytoplasmic Ca2+. Following the Ca2+ rise, the reuptake of Ca2+ into intracellular stores or efflux of Ca2+ out of the egg drive the return of cytoplasmic Ca2+ back to baseline levels. The molecular mediators of these Ca2+ fluxes in different organisms include Ca2+ release channels, uptake channels, exchangers and pumps. The functions of these mediators are regulated by their particular activating mechanisms but also by alterations in their expression and spatial organization. We discuss here the molecular basis for modulation of Ca2+ signalling at fertilization, highlighting differences across several animal phyla, and we mention key areas where questions remain.Calcium (Ca In this treatise, he states regarding fertilization:It [fertilization] is the central decisive event in the genesis of all sexually produced animals and plants. Thus from one point of view it envisages the entire problem of sex; from another point of view it constitutes the basis of all development and inheritance. The elements that unite are single cells, each usually incapable, under natural conditions, of continued existence or development—on the point of death; but by their union a rejuvenated individual is formed which constitutes a link in the eternal procession of life by virtue of its power of reproduction. How the terminally differentiated gametes interact with each other at fertilization to initiate the development of a new organism is a question that has fascinated scientists for over a century. The earliest studies of fertilization, largely performed in marine animals, were summarized in 1919 by Frank Lillie in 2+) across the egg  and Ca2+ imaging, we now know that a Ca2+ rise signals the initiation of animal development with no exceptions with cross-phyla sampling and even in several species of flowering plants [2+ rises at fertilization varies widely. Fertilization-induced Ca2+ changes depend on a large number of variables including exactly how the Ca2+ rise is initiated and how the newly formed zygote responds to this Ca2+ rise and perhaps to other signalling pathways triggered at fertilization. The collective set of responses, including the Ca2+ rise, that is required for the oocyte to begin development into an embryo is termed ‘egg activation’. Here, we will review the generation and modulation of Ca2+ signals during egg activation, focusing mainly on knowledge gained from intensive studies of mammalian fertilization over the past 30 years. However, we will also draw from studies of non-mammalian animals to illustrate additional mechanisms of generating and modulating the Ca2+ signals that initiate embryonic development.Despite this early scientific interest in fertilization, it was not until almost 60 years later that Lionel Jaffe and co-workers discovered that the sperm initiates a wave of calcium Ca+ across  in 1928 ,4. These protein . Similarg plants ,8. Howev2+ is a ubiquitous second messenger responsible for numerous cellular responses to external stimuli. Its ubiquitous nature implies that Ca2+ signals must be tightly regulated and that additional information must be encoded in the localization, amplitude and timing of Ca2+ release events. In this way, different stimuli can be properly interpreted by the cell and result in the necessary downstream responses. For example, basal cytoplasmic Ca2+ levels maintained by constitutive Ca2+ release from the endoplasmic reticulum (ER) suppress autophagy whereas higher levels of cytoplasmic Ca2+ promote autophagy [2+ signals promote contraction whereas nuclear Ca2+ pulses activate transcription of specific genes [2+ influx across the plasma membrane drives specific transcriptional responses not induced by global cytoplasmic Ca2+ rises [2+ under the control of a ‘Ca2+ signalling toolkit’ composed of Ca2+-mobilizing signals, channels that regulate Ca2+ influx into the cytoplasm or release from intracellular stores, and pumps and exchangers that remove Ca2+ from the cytoplasm [2+ signals.  Thi2.2+ signalling is feasible due to the presence of tightly regulated and localized Ca2+ gradients in the cell. A simplified schematic showing the relationships between the main modulators of cellular Ca2+ signalling is shown in 2+ is available to cells from the extracellular environment, which has very high (approx. 2 mM) Ca2+ levels relative to cytoplasmic Ca2+ that is normally in the 100 nM range. The entry of extracellular Ca2+ into the cell is tightly regulated by Ca2+-permeable channels on the plasma membrane that are sensitive to a variety of stimuli. For example, voltage-gated Ca2+ channels open in response to changes in membrane potential, whereas the large family of transient receptor potential (TRP) channels responds to diverse stimuli including temperature, stretch and osmolarity [2+ entry’ (SOCE) is a cellular mechanism to replenish depleted ER Ca2+ stores with Ca2+ from the extracellular environment. When ER stores are depleted, ER-resident STIM proteins oligomerize and interact with ORAI plasma membrane Ca2+ channels to stimulate Ca2+ influx [2+ into the cytoplasm due to the large concentration gradient from extracellular to intracellular cytoplasmic compartments.Camolarity ,17. ‘Sto+ influx . When op2+ is largely sequestered within the ER (or sarcoplasmic reticulum of muscle cells), but additional intracellular Ca2+ stores include endolysosomes and mitochondria. Peripheral tubular regions of the ER contain membrane contact sites with the plasma membrane and many organelles including lysosomes and mitochondria [2+ and other molecules between the two compartments. Ca2+ is released from ER stores mainly through two channels: the inositol-1,4,5-trisphosphate (IP3) receptor and the ryanodine receptor. Both of these channels are activated by Ca2+ itself, which can induce an autocatalytic process termed ‘Ca2+-induced Ca2+ release’ and lead to the propagation of waves of Ca2+ across a cell. IP3 and sn-1,2 diacylglycerol (DAG) are generated by the action of phosphoinositide-specific phospholipase C (PLC) enzymes on phosphatidyl inositol-4,5-bisphosphate (PIP2), often in response to ligand–receptor interactions at the plasma membrane. IP3 binding to IP3 receptors leads to Ca2+ release from ER stores in part by sensitizing the IP3 receptor to Ca2+. DAG can directly impact canonical TRP channel activity and indirectly influence Ca2+ signals by altering protein kinase C activity [2+ mobilizing signals, independent of IP3-mediated signalling, are generated from nicotinamide-adenine dinucleotide (NAD) and NADP: cyclic ADP ribose (cADPR) and nicotinic acid dinucleotide phosphate (NAADP) [2+, resulting in Ca2+ release from ER stores. NAADP, in contrast, signals to two-pore channels in lysosomes and other acidic organelles, resulting in Ca2+ release from those membranous stores. Mitochondria, which normally have relatively low Ca2+ stores, release Ca2+ through the sodium–Ca2+ exchanger, NCLX [Intracellular Cachondria . At thesactivity ,21. Two  (NAADP) . cADPR ser, NCLX ,24.2+ must be removed rapidly to terminate signalling and return cytoplasmic levels to baseline. Cytoplasmic Ca2+ removal is accomplished through the combination of extrusion from the cell and reuptake into intracellular stores. Extrusion across the plasma membrane is mediated by plasma membrane Ca2+ ATPase (PMCA) pumps and sodium–Ca2+ exchangers (NCX). Perhaps the most important mechanism of reuptake into intracellular stores is through sarco-ER Ca2+ ATPase (SERCA) pumps, which actively transport Ca2+ across a steep concentration gradient into the ER. Lysosomes probably also have Ca2+ uptake mechanisms, but the molecular basis for this function is not known [2+ through the mitochondrial Ca2+ uniporter and, under conditions of high cytoplasmic Ca2+, through the voltage-dependent anion channel [2+ signals determine the timing and localization of Ca2+ release, reuptake and extrusion. The end result is extraordinary variability in the final Ca2+ signals experienced by different regions of the cell, explaining the wide variety of distinct signalling pathways that use Ca2+ as a second messenger.Once released into the cytoplasm, Caot known . Mitocho channel . Alterat3.Caenorhabditis elegans and some molluscs, or meiosis may arrest again at various stages to coordinate with the timing of fertilization [In all animals, oocytes are arrested in prophase of meiosis I while they undergo growth and differentiation. In response to hormonal or developmental signals, oocytes undergo nuclear maturation, which entails completion of two rounds of chromosome segregation, and cytoplasmic maturation, which is a general term for the changes in cytoplasmic contents and organization necessary to support fertilization and embryo development. The oocyte's prophase I nucleus is termed the ‘germinal vesicle’ (GV) and we refer to oocytes in this meiotic stage as GV oocytes. Following the maturation signal, the GV breaks down (GVBD) and meiosis resumes. Subsequent stages of meiosis may continue without interruption, such as in lization . In most2+ signalling toolkit, their capacity to elicit Ca2+ responses is reduced compared with eggs, indicating that the ability to mount a robust Ca2+ response is acquired during oocyte maturation. This concept was first discovered in starfish oocytes, in which Ca2+ release in response to fertilization is far lower in immature oocytes relative to mature eggs, despite having similar Ca2+ stores [in vitro display Ca2+ oscillations of lower amplitude, frequency and persistence than those observed in eggs [2+ release in response to IP3 or PLCζ injection is reduced in GV oocytes compared with eggs [2+ response: increase in Ca2+ stores, ER redistribution and increase in sensitivity to IP3, probably due to upregulation and phosphorylation of IP3 receptors. Ca2+ stores increase about four-fold during mouse meiotic maturation [2+ leak out of the ER through the IP3 receptor [2+ store commences.Even though GV oocytes possess a Ca+ stores . In the  in eggs –29. Simiith eggs ,30,31. Sturation . Interesreceptor . This le2+ oscillatory behaviour following fertilization [2+/calmodulin-dependent protein kinase II (CaMKII), protein kinase A (PKA) and protein kinase C (PKC) [et al. showed that MPM2 immunoreactivity of IP3R1 was barely detectable at the GV stage, increased sharply around GVBD, reached peak levels at MI and MII, and decreased again after fertilization [ζ injection or caged IP3 expression [An increase in sensitivity to IP3 during oocyte maturation was first described in starfish oocytes and later demonstrated in hamster and mouse oocytes –28. In tlization ,34–36. Dlization ,31,37. H C (PKC) . Using tlization . This tipression . These f2+ regulatory protein whose amount increases during maturation and has an important function in eggs. Regulator of G-protein signalling 2 (RGS2) is a protein that blocks the activity of both Gαs and Gαq proteins, thus inhibiting heterotrimeric G-protein-coupled receptor pathways that frequently trigger Ca2+ release. RGS2 protein levels are extremely low in GV oocytes, but the mRNA is recruited for translation during oocyte maturation, such that RGS2 levels increase approximately 20-fold [2+ release in eggs, which helps to prevent spontaneous egg activation events from beginning inappropriately prior to fertilization.The IP3R is not the only Ca 20-fold . RGS2 pr2+ store, ER redistribution during oocyte maturation has been extensively studied and is correlated with the acquisition of a robust Ca2+ response. Using the fluorescent lipophilic dye DiI to label ER membranes, studies in starfish, marine nemertean worm, frog, hamster, human, mouse and plant oocytes demonstrated changes in ER organization during oocyte maturation [2+ store and its releasing channel in proximity to the site of sperm–egg fusion, which is where the propagating Ca2+ wave starts at fertilization. The mechanism of this ER redistribution is a multi-step process driven by both microtubules and microfilaments [Padi6- and Nlrp5-deficient oocytes, which lack these structures, fail to form cortical clusters [2+ oscillations are impaired in Nlrp5 null eggs [Xenopus oocytes, the ER-rich structures found in immature oocytes have been characterized as annulate lamellae (AL). AL are rich in IP3 receptor content, but paradoxically show minimal Ca2+ release activity, as opposed to ER patches in eggs, from which most Ca2+ is released in response to IP3. This finding suggests that AL are a functional Ca2+ store with attenuated IP3 receptor activity [During the course of oocyte maturation, there is a reorganization of several organelles, including the ER, Golgi apparatus and mitochondria. Because the ER is the main cellular Caturation –47. In mturation . During turation . The eggturation ,48. The clusters ,50. Furtactivity . Whetheractivity .2+ store. Golgi membranes contain functional IP3 receptors [2+ release during egg activation is unknown. It is also unclear if there is an important role for the reorganization of the Golgi apparatus during oocyte maturation, or whether it is simply a response to major ER rearrangements or changing trafficking demands. One possible role for the Golgi fragments is serving as an intracellular source of PIP2 for hydrolysis by PLCζ at fertilization. The amount of vesicular PIP2 increases during oocyte maturation and, interestingly, the localization pattern of both PIP2 and PLCζ suggests that the vesicles could be part of the Golgi apparatus [The Golgi apparatus of mouse, rhesus monkey and bovine GV oocytes comprises a series of cytoplasmic stacks, or ‘mini-Golgis’, more abundant in the interior than in the cortical region of the cell ,54. Despeceptors , but thepparatus .2+ oscillations, at least in part by providing the ATP required for Ca2+ pumps, these changes in mitochondrial localization, clustering and ATP output during oocyte maturation may be necessary to elicit a robust Ca2+ response at fertilization.Mitochondria also undergo redistribution during oocyte maturation. At GVBD, they form a dense ring in the perinuclear area, followed by dispersion throughout the cytoplasm and formation of a new mitochondrial ring around the meiotic spindle –60. By t4.2+ signalling is a universal hallmark of egg activation in all sexually reproducing species studied thus far. However, sperm-induced Ca2+ signals are significantly different across multiple phyla [2+ dynamics during egg activation is schematized in 2+ pattern takes the form of a single or a few waves. One exception is the marine worm Urechis caupo, in which the sperm triggers entry of extracellular Ca2+ uniformly around the egg cortex and no Ca2+ waves occur [C. elegans, a local cytoplasmic Ca2+ increase occurs immediately after sperm–egg fusion, and a single Ca2+ wave propagates from the sperm entry site across the entire egg [2+ wave generally lasts from one to several minutes. Drosophila eggs also have a single Ca2+ wave; however, it is initiated from the egg pole through mechanical pressure and moves towards the centre of the oocyte and finally to the entire egg [2+ wave that spreads across a small portion of the surface of the egg. Collectively, the multiple Ca2+ waves triggered around the whole egg ensure a global and long-lasting intracellular Ca2+ increase necessary for egg activation [2+ pattern is a series of oscillations that travel in a wave-like fashion across the egg and last for minutes to hours. For example, fertilized marine nemertean worm and marine bivalve eggs display multiple oscillations that can persist for 30–60 min [2+ oscillations after sperm–egg fusion, but these persist for several hours [As mentioned previously, Cale phyla . The dives occur . In medatire egg ,64,66,70tire egg . In spectivation ,72. Anot0–60 min –75. Simial hours ,77.Figu2+ dynamics vary widely among species, the Ca2+ oscillatory pattern is stereotypical for mammals. Shortly after sperm–egg fusion, the egg intracellular Ca2+ concentration rises more than 10-fold and persists close to this level for several minutes before returning to baseline. This initial relatively long Ca2+ transient is considered the main Ca2+ signal responsible for egg activation. The characteristic shape of this first Ca2+ transient has been well documented in different mammalian species using ratiometric Ca2+ imaging with epifluorescence signal detection [2+ concentration rises slowly at first and then rises rapidly to a peak, frequently followed by several short low-amplitude increases from the peak. The Ca2+ level then gradually falls over the course of several minutes before dropping rapidly back to baseline as cytoplasmic Ca2+ is cleared. The first transient is followed by a series of much shorter Ca2+ transients that are also characterized by an initial slow rise in Ca2+, a rapid rise to a peak that is usually lower than that of the first transient, and then rapid return to baseline [2+ oscillations persist for several hours, until around the time of pronucleus formation. The frequency of Ca2+ oscillations appears to be a species-specific characteristic; however, it is possible that differences in media composition rather than inherent species differences influence oscillation frequency.Although Caetection ,78–80. Cbaseline . In mamm2+ homeostasis orchestrate early and late events of egg activation. Artificial modulation of the Ca2+ oscillatory pattern by electropermeabilization revealed a correlation between the total number of Ca2+ transients applied and distinct cellular events of mouse egg activation [2+ transient, whereas more than four transients are necessary to induce cell cycle resumption and polar body emission. Furthermore, robust pronuclear development, which is a later event of egg activation, requires additional electropermeabilization-induced Ca2+ transients. Studies in rabbit and mouse eggs concluded that the different egg activation events rely on a cumulative effect of Ca2+ input over downstream effectors, rather than a specific number or frequency of Ca2+ signals [2+ pattern for activation, if the total summation of Ca2+ stimuli is sufficient to trigger egg activation.Long-lasting fertilization-associated changes in Cativation . Thus, c signals –85. Ther2+ signalling during or immediately after fertilization of mouse eggs results in alterations in the pre- and post-implantation developmental programs and even postnatal growth rate.  In criptome . This fipatterns . Moreove to term . Similaron rates . It is ncontrols . These f in vivo . Therefoactivity .in vitro studies recapitulate the complex regulation of in vivo fertilization, in terms of the need for a specific Ca2+ oscillatory pattern for developmental success. Doubts arose after it was reported that several different genetic mouse models in which eggs show a very abnormal pattern of sperm-induced Ca2+ oscillations after IVF were still capable of producing offspring when fertilization occurred in vivo [2+ oscillations to activate eggs effectively in vivo. Further studies are needed to define the fertilization-associated Ca2+ oscillatory patterns in vivo, within the highly specialized environment of the oviduct, and to determine whether alterations in Ca2+ signalling in vivo have similar detrimental effects on offspring health as artificial disruption in vitro.Despite advances in our understanding, it remains unclear whether these findings reported mainly from  in vivo ,91,92. T5.5.1.2+ mobilizing signal at fertilization and was the first identified. Early biochemical studies in sea urchins documented a rapid rise in both triphosphoinositides and diphosphoinositides that occurred after insemination but prior to evidence of Ca2+ release, suggesting that IP3 mediates Ca2+ release as had been shown previously in somatic cells [2+ concentration [2+ release occurs at fertilization across multiple phyla [IP3 is the major Caic cells . This idntration . Subsequle phyla –98.2+ release, some species use alternative or additional mechanisms. In sea urchins, cADPR serves as a redundant mechanism for Ca2+ release at fertilization, presumably by activating the ryanodine receptor [2+ release at fertilization [2+ entry from the extracellular milieu serves as a mechanism to increase cytoplasmic Ca2+ in echinoderms, molluscs and worms. In starfish, Ca2+ entry is a response to sperm-induced activation of a voltage-gated Ca2+ channel; this interaction results in a ‘cortical flash’ of Ca2+ [2+ release contribute to the cortical flash [2+ influx, and not intracellular stores, to provide the fertilization Ca2+ signal [C. elegans, Ca2+ enters the egg through TRP channels located in the sperm plasma membrane following sperm–egg fusion [2+ entry as was first proposed by Jaffe [2+ entry is responsible for the rapid local rise in Ca2+ that precedes the global wave that crosses the egg, analogous to the cortical flash in starfish, but is not essential for the occurrence of the later global Ca2+ wave or for embryogenesis.Besides IP3-induced Careceptor ,99. Therlization –102. Ca2 of Ca2+ ,104. In al flash . Limpets+ signal . In C. eg fusion . In thisby Jaffe . This mo2+ wave responsible for activating Drosophila eggs is initiated from extracellular Ca2+ sources in response to the mechanical stimulation of egg plasma membrane TRP channels during ovulation [2+ release [Sicyonia shrimp induces a Ca2+ wave in the absence of sperm [2+ wave is initiated downstream of magnesium ions in seawater, but the molecular mechanism underlying Ca2+ entry is unknown [2+ wave from the fertilizing sperm creates a requirement for highly efficient single sperm entry soon after egg activation to ensure that diploid embryonic development begins synchronously.Some species undergo physiological egg activation in the absence of sperm. For example, the Cavulation ,108. Theof sperm ,110. In 5.2.2+ release at fertilization in most species studied, but the specific PLC used varies. There are six families of PLC enzymes in animals: PLCβ, PLCγ, PLCδ, PLCε, PLCζ and PLCη [2+-binding EF hand motifs. Hence, they all carry out PIP2 hydrolysis to generate IP3 and DAG and can be modulated by changes in Ca2+ levels.Generation of IP3 by PLC-mediated hydrolysis of PIP2 is essential for Caand PLCη . Each PL2+ release were performed in the starfish Asterina miniata. Carroll and colleagues demonstrated that src homology-2 (SH2) domain-mediated inhibition of PLCγ activity prevents the sperm-induced Ca2+ wave that follows the cortical flash [Ciona intestinalis revealed that PLCγ mediates Ca2+ release in these marine invertebrates as well [2+ wave. In addition, these findings suggested that a tyrosine kinase functions upstream of PLCγ activity at fertilization given that this is the most common mechanism of activating PLCγ [Xenopus demonstrated that src family tyrosine kinases stimulate Ca2+ release at fertilization by activating PLCγ [γ/IP3 pathway to initiate Ca2+ release appears to be a common mechanism among external fertilizing species. However, in mouse, SH2-domain-mediated inhibition of PLCγ activity has no impact on sperm-induced Ca2+ oscillations, indicating that either PLCγ is not involved or that it must be activated by an alternative mechanism [The first experiments to identify a specific PLC that was activated at fertilization to generate the IP3 responsible for Caal flash . Similar as well . These fing PLCγ . Furthering PLCγ ,114. Henechanism .2+ release following sperm–egg plasma membrane fusion at fertilization was first proposed by Whitaker & Irvine [2+-induced exocytosis of cortical granules [2+-dependent egg activation events in distinct phyla [2+ release at fertilization, sperm–egg fusion precedes the increase in cytoplasmic Ca2+ in sea urchin, frog and mouse [ζ, was cloned from a mouse spermatid cDNA library and identified convincingly as the protein in sperm extracts capable of activating eggs [ζ caused Ca2+ oscillatory patterns very similar to those observed at fertilization. Importantly, removal of PLCζ from hamster sperm extracts by an immunodepletion strategy caused the loss of the extract's Ca2+ releasing activity following microinjection into mouse eggs. Finally, mutation of a critical catalytic site residue abrogated this activity, indicating that it was the PLCζ enzymatic activity that was responsible for Ca2+ release. These studies effectively demonstrated that PLCζ was the critical component in microinjected sperm extracts that induces Ca2+ release, but left open the question of whether or not PLCζ was important for fertilization-induced Ca2+ release.The hypothesis that sperm introduce a PLC activity into the egg that is responsible for PIP2 hydrolysis and Ca& Irvine . This idgranules . Similarct phyla –119. Connd mouse ,120–122.ing eggs . Microinin vivo model to describe a role for PLCζ at fertilization was generated using a mouse transgenic RNA interference (RNAi) knockdown approach that resulted in an approximately 40% reduction in sperm PLCζ protein levels [in vitro by these sperm had abnormal patterns of Ca2+ oscillations that terminated prematurely. Subsequent experiments using human sperm lacking PLCζ obtained from infertile males demonstrated that the sperm failed to induce Ca2+ release when injected into mouse eggs [PLCZ1 were identified that were associated with a case of male infertility refractory to ICSI [2+ oscillation-inducing ability in mouse eggs [PLCZ1 mutations associated with human egg activation failure have since been identified [Plcz1 models confirmed that PLCζ is essential for normal Ca2+ oscillations following fertilization [2+ oscillation phenotype in one of these models revealed that eggs fertilized in vitro by a single sperm exhibited approximately 3 Ca2+ transients on average, but that the onset of these transients was delayed by approximately 45–60 min relative to eggs fertilized with wild-type sperm [ζ did not induce any Ca2+ transients following ICSI, suggesting that events associated with sperm–egg plasma membrane fusion were essential for this PLCζ-independent Ca2+-releasing activity. The molecular basis for this additional egg activation mechanism is unknown.The first n levels . Eggs feuse eggs . This fi to ICSI ,127. Botuse eggs . Additioentified –132. Thelization ,92. Surpζ, the smallest PLC identified to date, is unique when compared with previously identified PLCs [ζ explains its ability to diffuse freely through the egg cytoplasm. Indeed, there is evidence that plasma membrane PIP2 does not serve as a substrate for PLCζ at fertilization [2+ oscillations induced by the fertilizing sperm [ζ with these vesicles appears to be mediated by basic residues in the XY-linker, EF-hand and C2 domains [ζ. The XY-linker in PLCζ is positively charged and its deletion causes a loss of enzymatic activity, suggesting that PLCζ is constitutively active [2+ is the only known activator of PLCζ, with an EC50 of approximately 50 nM and 70% maximal activity at 100 nM [ζ is almost maximally active at the basal intracellular Ca2+ levels present in the egg cytoplasm. By contrast, other PLCs are activated by approximately 10-fold higher Ca2+ levels [PLCied PLCs . It lackied PLCs . Becauselization ,133. In ng sperm ,134. The domains ,135–137. domains . This isy active . Ca2+ ist 100 nM . Hence, + levels .ζ is responsible for the PLC activity that initiates Ca2+ release during egg activation, an open question is whether or not endogenous egg PLCs contribute to the Ca2+ oscillatory pattern by generating additional IP3-mediated Ca2+ release. On somatic cell membranes, ligand-G-protein-coupled receptor interactions frequently stimulate PLCβ isoform-mediated generation of IP3 and DAG through the activity of heterotrimeric G-protein α subunits in the Gαq family [β. Support for this mechanism came from the observation that overexpression of the Gαq-coupled m1 muscarinic receptor in mouse eggs followed by exposure to its cognate ligand, acetylcholine, caused egg activation events downstream of cytoplasmic Ca2+ elevation [αq could cause complete egg activation, sperm did not require this activity to successfully activate eggs, indicating that Gαq-mediated PLCβ stimulation was not required for Ca2+ release at fertilization [β was also tested using a knockdown strategy to generate mice carrying eggs with decreased levels of PLCβ1 [β1 caused a significant decrease in the amplitude of sperm-induced Ca2+ transients, but did not impact other characteristics of the oscillatory pattern, suggesting a contributory but not major role for PLCβ1. An interesting recent study took advantage of the development of a new fluorescent IP3 sensor to monitor both Ca2+ and IP3 concurrently in fertilized mouse eggs [2+ oscillations following fertilization, Ca2+ activates PLC to generate IP3 peaks that follow each Ca2+ transient. These findings further support the idea that endogenous egg PLCs are active at fertilization and impact the overall Ca2+ signals experienced by the early embryo, but which PLC, if any, has yet to be determined.Although PLCq family . By anallevation ,141. Howlization . The rolof PLCβ1 . Reductiuse eggs . Using t5.3.2+ from the ER in response to IP3 and Ca2+ binding. It is composed of large subunits that assemble as tetramers to generate a greater than 1 MDa Ca2+ channel mainly present in ER membranes. Only a small portion of the IP3 receptor extends into the ER lumen; the majority extends into the cytoplasm, giving it a square mushroom-shaped structure that is evident by cryo-electron microscopy [2+ binding, which has a biphasic impact on channel opening [2+ levels promote channel opening, whereas high (greater than 300 nM) Ca2+ concentrations inhibit channel opening. Hence, Ca2+ released by channel opening feeds back on the IP3 receptor to promote closing, resulting in intermittent Ca2+ release events from single receptors. When IP3 receptors are enriched in specific locations, this feedback system leads to localized Ca2+-induced Ca2+ release as nearby receptors open in response to Ca2+ released by others. Finally, as sufficient Ca2+ is released to enable diffusion to more distant sites, distal IP3 receptors open. When supported by Ca2+-induced activation of PLC to generate additional IP3, this process can eventually lead to the formation of regenerative Ca2+ waves that travel across the cell . Th opening –154. Hen2+ through the IP3 receptor is essential at fertilization for most animals studied. This was first demonstrated in hamster eggs, in which microinjection of a function-blocking monoclonal antibody that inhibits Ca2+ release from the IP3 receptor prevented sperm-induced Ca2+ oscillations following fertilization [2+ release at fertilization was demonstrated using an ‘IP3 sponge’ that prevents receptor activation by absorbing IP3 [2+ through the related ER-resident ryanodine receptor is also necessary for a full Ca2+ release response [2+ release responsible for the fertilization-associated Ca2+ wave, but Ca2+ released through the ryanodine receptor promotes post-fertilization plasma membrane insertion events that do not occur when only the IP3 receptor is active [2+ release or other egg activation events at fertilization in mammals [The release of Calization . Similarlization . In starbing IP3 . Althougresponse ,104. In s active . Althoug mammals –161.Xenopus, Drosophila, C. elegans and starfish, appear to have a single IP3 receptor isoform [2+ release functions in response to IP3 and Ca2+, there are differences in their regulatory properties. For example, IP3R2 has a higher affinity for IP3 than IP3R1, and IP3R1 and IP3R2 are activated when phosphorylated by PKA, but IP3R3 is not [2+ release activity in mammalian eggs as first demonstrated in hamster eggs by Miyazaki et al. [Many animals, including  isoform ,162–164. isoform . The IP3 isoform . Althoug3 is not –169. IP33 is not ,170. In 3 is not ,36,170. i et al. .2+ signalling. In Xenopus oocytes, IP3 receptor quantity changes minimally during maturation but there are still significant differences in IP3 receptor-mediated Ca2+ release events between oocytes and eggs [An important consideration regarding the regulation of IP3 receptor activity is the particular ER subdomains where the receptor is localized. As mentioned above, IP3 receptor quantity, localization and phosphorylation status change during oocyte maturation in mammals and these changes probably impact Caand eggs ,163. It and eggs . In mamm5.4.2+ in response to stimulation by NAADP. These channels were first identified based on their similarities to voltage-gated Ca2+ channels and TRP channels [2+ from reserve (yolk) granules, a cellular compartment distinct from the ER and a functional equivalent of lysosomes [2+ are released from endolysosomal stores in response to NAADP; however, these Ca2+ release events can then trigger Ca2+-induced Ca2+ release mediated by ER-associated IP3 and/or ryanodine receptors [2+ release from ER stores can promote Ca2+ release from acidic stores through retrograde signalling [Two-pore channels, which localize to acidic organelles of the endolysosomal system, release Cachannels . Their iysosomes . The resysosomes . More reysosomes . It is leceptors ,174. Likgnalling . This crgnalling ,176.5.5.2+-ATPase (SERCA) is a P-type Ca2+ pump located in ER membranes that transports Ca2+ against a concentration gradient from the cytoplasm into the ER lumen. In mammals, it is encoded by three genes (Atp2a1–3) that generate the three main isoforms (SERCA1–3), but because the transcripts are subject to alternative splicing, they give rise to 11 SERCA isoforms [2+, and regulation. SERCA1a and 1b are expressed in adult and foetal fast-twitch skeletal muscle, respectively. SERCA2a is expressed in cardiac and slow-twitch skeletal muscle, whereas SERCA2b is ubiquitously expressed and considered the housekeeping SERCA isoform. SERCA3 isoforms are found in certain non-muscle cells, usually co-expressed with SERCA2b. The functional organization of SERCA proteins, like all Ca2+-ATPases, is composed of a transmembrane domain containing 10 membrane-spanning helices and three cytoplasmic domains: the actuator, phosphorylation and nucleotide-binding domains. The SERCA pump contains two Ca2+-binding sites in the transmembrane domain and hence transports two Ca2+ ions per ATP hydrolyzed. SERCA pumps can be inhibited by general P-type ATPase inhibitors, such as La3+ and orthovanadate, and by the specific inhibitor thapsigargin [The sarco-ER Caisoforms . These isigargin .Xenopus oocytes, endogenous SERCA2 is found in cortical clusters [2+ oscillations of higher frequency and shorter duration, indicating that SERCA isoforms probably are important in Ca2+ regulation at fertilization in frogs [2+ oscillations following fertilization was demonstrated by showing that thapsigargin treatment severely reduces the magnitude and duration of the first Ca2+ transient and decreases persistence of oscillations [2+ stores after depletion following fertilization.SERCA2 isoforms are the main variants present during fertilization. In clusters . Exogenoin frogs . In mousin frogs . A role llations . Whereasllations . This lo5.6.2+ pump that transports excess intracellular Ca2+ outside of the cell to maintain homeostasis. It has high affinity for Ca2+ and low transport capacity, and it exchanges Ca2+ for H+. Its domain structure is similar to other P-type pumps (one transmembrane and three cytoplasmic domains), but it also contains a calmodulin (CaM)-binding site in its C-terminus that functions as an auto-inhibitory domain. Under resting conditions, this domain folds into the ATP-binding site, inhibiting the pump. When intracellular Ca2+ increases, Ca2+-bound CaM interacts with the CaM-binding site, releasing the auto-inhibitory conformation of the pump and restoring activity [2+ responses [ζ is located in intracellular vesicles rather than in the plasma membrane.The PMCA is a P-type Caactivity ,178. PMCactivity ,183. Amoesponses . It remaAtp2b1–4) that are alternatively spliced to generate around 30 isoforms. PMCA1 and PMCA4 are ubiquitously expressed and considered the housekeeping PMCAs. PMCA2 is expressed in the nervous system and mammary gland, whereas PMCA3 is expressed in the nervous system [Xenopus oocytes and eggs, the presence and localization of PMCA was demonstrated using an antibody that recognizes all four mammalian PMCA isoforms [2+ oscillations in which the first transient has higher amplitude and longer duration, indicating a role for PMCA in extruding excess Ca2+ after fertilization [2+ oscillations triggered by PLCζ injection can be sustained in the absence of extracellular Ca2+ if Ca2+ efflux through PMCA is blocked by 5 mM Gd3+ [Atp2b1 is the most abundant isoform, followed by Atp2b3, while Atp2b2 and Atp2b4 are nearly undetectable [Mammalian PMCA is encoded by four genes . This transporter, present in the plasma membrane of most cells, exchanges three Na+ for one Ca2+ and the directionality of the fluxes depends on the membrane potential and the concentration gradients of these cations [2+-binding regulatory sites reside. In mammals, NCX is encoded by three genes, Ncx1–3, with Ncx1 and 3 undergoing alternative splicing. NCX1 is ubiquitously expressed, NCX2 is expressed in the nervous system, and NCX3 is expressed in the brain and skeletal muscle [2+ efflux varies depending on the tissue; it is essential in the heart to extrude Ca2+ from myocytes during each cardiac cycle, whereas it does not play a major role in the liver [Ca cations . NCX conl muscle . The relhe liver .+ causes the NCX to function in the reverse mode, leading to Ca2+ influx. This approach has been used to infer the presence of NCX in hamster, mouse, rat and Xenopus oocytes [Xenopus oocytes, further characterization demonstrated that both NCX1 and NCX3 are expressed in the plasma membrane and are more abundant in the animal hemisphere [+ depletion, as well as Na+ ionophore and Ca2+ addition after incubation in Ca2+-free medium, demonstrated that NCX is present in oocytes and eggs. However, the changes in Ca2+ fluxes observed after manipulating Na+ gradients are modest, and oocytes can recover from these perturbations, suggesting that NCX does not play an important role under physiological conditions [Removal of extracellular Na oocytes –195. In nditions .2+ efflux in metaphase II eggs compared with GV oocytes. Mouse oocytes respond to an increase in intracellular Na+ or a depletion of extracellular Na+ by increasing Ca2+ influx, due to the NCX ‘reverse mode’ activity. Mouse eggs, on the other hand, fail to respond to these changes in Na+ gradients [Xenopus, PMCA is endocytosed during oocyte maturation, leading to depletion of this ATPase from the plasma membrane and a reduction in Ca2+ efflux [2+ efflux during oocyte maturation is another mechanism to maximize Ca2+ responses at fertilization.Both PMCA and NCX undergo changes during oocyte maturation that suggest a decrease in Caradients . The rea+ efflux . Whether5.7.2+ influx occurs at fertilization across numerous species. For example, fertilization of sea urchin and marine worm eggs results in a dramatic increase in Ca2+ influx within a few minutes as indicated by radiolabeled Ca2+ uptake [2+ concentrations change the frequency of periodic hyperpolarizing responses, suggesting that Ca2+ influx modulates the frequency of the intracellular Ca2+ release events responsible for hyperpolarization [2+, fertilization-induced Ca2+ oscillations rapidly cease due to the depletion of ER Ca2+ stores [2+ influx in mouse eggs is required for second polar body emission, suggesting that cortical Ca2+ signals downstream of Ca2+ influx are essential for complete egg activation [We have long been aware that Ca+ uptake ,198. In rization ,191. In + stores ,191. In tivation .2+-permeable channels on the plasma membrane that when open allow Ca2+ to transit from the extracellular milieu down an approximately 10 000-fold concentration gradient into the cytosol. These channels are activated by a wide variety of signals. The voltage-gated Ca2+ channels open in response to changes in membrane potential and are best known for their roles in excitable cells such as neurons and cardiomyocytes. The TRP channels are ubiquitously expressed, and most are involved in transmitting sensory inputs or environmental signals. The TRP channels are permeable to multiple cations in addition to Ca2+, with variable selectivity toward different cations, and are functional at resting membrane potentials in non-excitable cells [2+ channels on the plasma membrane are activated in response to depletion of intracellular Ca2+ stores, often following PLC-mediated activation of Ca2+ release through the IP3 receptor. There is experimental evidence for a role for each of these three channel classes in supporting Ca2+ influx necessary for successful egg activation, though the evidence for SOCE is very limited.There are numerous Cale cells . Ubiquit2+ store depletion, STIM1 and STIM2, which are ubiquitously expressed single transmembrane proteins with Ca2+-binding EF hand domains in the ER lumen. In response to ER store depletion, Ca2+ dissociates from the STIM proteins, which then oligomerize and move toward ER-plasma membrane contact sites. STIM oligomers form large clusters, or punctae, that directly interact with plasma membrane ORAI channels, stimulating Ca2+ entry. Because depletion of ER Ca2+ stores occurs in the many species that depend on IP3-mediated Ca2+ release at fertilization, SOCE appears to be an obvious mechanism to replenish ER stores. However, SOCE is inactivated in mammalian cells during mitosis [Xenopus it is inactive during meiosis [2+ stores in mature oocytes prior to fertilization. Because low ER Ca2+ stores before fertilization would be anticipated to result in inhibition of Ca2+ oscillatory behaviour following fertilization, these studies were not conclusive. In an effort to definitively determine whether SOCE was required to replenish ER stores at fertilization, a mouse knockout approach was used to generate eggs lacking both STIM1 and STIM2; Orai1-null eggs were also tested [Stim1/Stim2 double knockout eggs had normal ER Ca2+ stores and no alterations in Ca2+ influx or Ca2+ oscillatory behaviour at fertilization relative to controls. Furthermore, eggs lacking ORAI1, the only ORAI channel expressed in mouse eggs, exhibit normal Ca2+ handling and homeostasis at fertilization. These findings clearly indicate that in the mouse, SOCE is not the Ca2+ influx mechanism responsible for refilling ER stores following fertilization. It remains possible that in other animals SOCE could be used.SOCE is well established as a major mechanism in somatic cells whereby ER stores are replenished following depletion ,200. Thi mitosis  and in X meiosis , suggest meiosis ,204, but meiosis ,205. Oth meiosis . Both of meiosis ,208. Unfo tested . Stim1/S2+ channels (CaV) are also important mediators of calcium influx. They are composed of a pore-forming α1 subunit associated with an intracellular β subunit, and extracellular α2 subunit bound by disulfide linkage to a transmembrane δ subunit, and sometimes a transmembrane γ subunit [α1 subunits, which are encoded by ten distinct genes in mammals. The voltage-gated Ca2+ channels are divided into three families based on their structure and function. The CaV1 channels are sensitive to large changes in membrane potential and tend to support long-lasting, large-conductance Ca2+ currents; hence, they are known as ‘L-type’ channels. The CaV2 channels are largely expressed only in brain regions and will not be discussed further here. The CaV3 channels are sensitive to small changes in membrane potential at negative voltages, are rapidly inactivated, and have transient kinetics leading to their characterization as ‘T-type’ channels. An interesting feature of T-type channels is that they can be open even in resting cells at a window of membrane potential between the activating and inactivating potentials; the resulting inward Ca2+ current is called ‘window’ current [Voltage-gated Ca subunit . Their d current .2+ channels in supporting Ca2+ influx following fertilization in different species. Many marine animals and amphibians use L-type Ca2+ channels for this purpose; their opening is triggered by a large change in membrane potential  that occurs in response to either sperm–egg binding or fusion [Pseudopotamilla occelata use L-type channels to generate the global Ca2+ wave responsible for resumption of meiosis, but not the initial sperm-induced Ca2+ increase [Mytilus edulis, the initial rise in Ca2+ following fertilization is dependent on Ca2+ influx through L-type channels, whereas internal Ca2+ stores provide later Ca2+ signals [2+ influx through L-type channels to provide the egg activation signal [V3.2, which is likely to be active through a window current mechanism [V3.2 have lower levels of Ca2+ influx during oocyte maturation and following fertilization. Furthermore, female mice whose eggs lack CaV3.2 have reduced litter sizes, suggesting that this channel is critical for efficient egg activation and development to term [V3.2 cannot be the only Ca2+ influx channel active at fertilization in the mouse because in at least some eggs lacking CaV3.2, Ca2+ oscillations can persist long-term following fertilization.There is evidence for both L-type and T-type Car fusion . For exaincrease . In the  signals . Limpet n signal ,212. Mamn signal ,214. Hown signal ,216. Theechanism . Mouse e to term . However2+ influx at fertilization. The TRP superfamily is encoded by a large number of distinct protein-coding genes: 28 in mice, 17 in C. elegans, and 13 in Drosophila [2+ influx at fertilization, at least in the mouse, because mice globally lacking all seven TRPC channels are fully fertile [2+ influx and egg activation [Trpv3-null mice are fertile, and their eggs have no alterations in sperm-induced Ca2+ oscillatory patterns, indicating that this channel is dispensable, though it could contribute to supporting Ca2+ influx at fertilization. TRPM7 is a constitutively active ion channel permeable to divalent cations including magnesium and Ca2+, but it can be activated by mechanical signals and inhibited by low millimolar magnesium levels [2+ influx. Chemical inhibition of these channels in fertilized one-cell embryos impairs development beyond the two-cell stage [2+ influx at fertilization, Trpm7 was conditionally deleted from mouse eggs using the Gdf9-cre transgene [2+ influx was lost. Furthermore, they had significantly blunted responses to alterations in extracellular Ca2+ and magnesium, and reduced Ca2+ oscillation frequency and persistence following fertilization. Although female mice carrying Trpm7-null eggs were fertile, their heterozygous offspring had abnormalities in weight variance and growth trajectories. These findings indicate that TRPM7 mediates spontaneous and SOCE-like Ca2+ influx, is largely responsible for the impact of extracellular Ca2+ and magnesium concentrations on Ca2+ influx, and is required to support Ca2+ influx following fertilization.In the past few years, attention has turned to the possible roles of TRP channels in supporting Caosophila . TRP chaosophila . It is u fertile . TRPV3 i fertile , suggesttivation . Howeverm levels ,223. TRPm levels . Functioll stage . To defiransgene . Eggs laV3.2 in supporting Ca2+ influx at fertilization, a double knockout mouse model was established to generate eggs lacking both CaV3.2 and TRPM7 [2+ stores and minimal Ca2+ influx following ER store depletion. During approximately the first hour following fertilization, the double knockout eggs had only one or two Ca2+ transients, a finding very similar to the oscillatory pattern observed in wild-type eggs cultured after fertilization in Ca2+-free medium. These findings indicate that CaV3.2 and TRPM7 are largely responsible for the Ca2+ influx needed to replenish ER stores and to support persistent sperm-induced oscillations. However, many of the double knockout eggs restarted their oscillations again after about an hour, suggesting that a new Ca2+ influx mechanism becomes active over time. The restart pattern did not occur when the double knockout eggs were fertilized by ICSI, indicating that the new influx mechanism depends on either sperm–egg plasma membrane interaction or fusion. Female mice carrying CaV3.2/TRPM7 double knockout eggs had reduced litter sizes explained by differences in implantation or post-implantation development and the offspring had increased weight variability relative to controls. These findings indicate that together, CaV3.2 and TRPM7 serve as essential mediators of Ca2+ influx following fertilization in mice, though additional channels such as TRPV3 could also contribute. Whether or not they serve a similar function in eggs of other mammals is not known.Given the previously established role of Cand TRPM7 . These e2+ influx at fertilization is conserved in insects and worms. As mentioned above, Drosophila egg activation is initiated by Ca2+ influx that occurs in response to mechanical pressure during ovulation, though propagation of the Ca2+ wave depends on IP3-mediated Ca2+ release [2+ influx is mediated by Trpm, the only Drosophila orthologue of mammalian TRPM7 [C. elegans, a sperm plasma membrane TRP channel, TRP-3, serves as a conduit for localized Ca2+ influx into the egg following sperm–egg fusion [2+ wave is delayed and abnormally shaped. Taken together, these findings indicate that TRP channels are essential modulators of Ca2+ signals required for the activation of development in both protostomes and deuterostomes. A schematic illustrating the Ca2+ release, reuptake and efflux mechanisms active in mouse eggs following fertilization is shown in There is new evidence that TRP channel function in Ca release ,226. It an TRPM7 . In C. eg fusion . In the 6.2+ levels. Although the importance of Ca2+ was not known at the time, Jacques Loeb found during a series of studies beginning in the late 1800's that placement of sea urchin eggs into hypertonic solutions resulted in the elevation of the fertilization envelope, an initial step of fertilization [2+ in these artificial processes.Eggs of numerous species can be activated in the absence of sperm, known as parthenogenetic activation, by artificially increasing cytoplasmic Calization . It was lization –230. Baslization . This bolization ,233. Mor2+ levels that artificially triggers cell cycle resumption and embryo development. We refer to these methods as ‘artificial egg activation’ (AEA) because they are often used in the presence of the paternal genome and therefore are not the same as parthenogenetic activation. Whereas some of these methods mimic the physiological repetitive Ca2+ increases observed in mammals, others simply induce a single large rise in Ca2+ that is sufficient to activate the downstream effectors necessary to complete egg activation. Finally, alternative methods completely bypass the Ca2+-dependent steps to induce AEA by activating directly the necessary downstream signalling pathways; these methods will not be discussed here as they are outside the scope of this review. Typical Ca2+ traces observed in mammalian eggs following the various methods of egg activation are shown in Many different methods are used to induce the rise in intracellular Ca6.1.Drosophila egg activation is initiated by mechanosensitive ion channels. These eggs can be artificially activated by osmotic swelling after exposure to hypotonic water or by applying hydrostatic pressure to mature eggs [2+ entry provoked after mechanical membrane disruption of frog eggs also results in AEA.Mechanical activation can be achieved efficiently in a small number of species. Under physiological conditions, ure eggs . Ca2+ en2+ from the extracellular space causes a transient rise in intracellular Ca2+. The advantage of this method is that the Ca2+ rises can be controlled completely based on the equipment settings. Traditionally, electrical pulse length was from milliseconds to microseconds, but these relatively long-duration electric pulses can open large pores that are irreversible and lead to cell death [2+ efflux from the ER and even induce spontaneous Ca2+ oscillations while maintaining the integrity of the plasma membrane [Electrostimulation has been used in several species to trigger egg activation ,83,236. ll death . New tecll death . In mousmembrane . This pr6.2.2+ transient to spontaneous Ca2+ oscillations. The characteristic responses serve as a reference for their classification  bind Ca2+ in a 1 : 1 ratio and facilitate its transport across the cell membrane. In somatic cells, exposure to calcimycin promotes Ca2+ efflux from intracellular stores as well as Ca2+ influx from the extracellular space [2+ influx [2+ followed by a slow decline over the course of several minutes. Ca2+ mobilization stops after drug washout, resulting in a transient effect, though additional ionophore treatments can result in additional Ca2+ increases in the same egg. Ca2+ ionophores are used in some human ART clinics to artificially activate eggs that fail to activate following ICSI. Although both ionophores function similarly, a direct comparison between ionomycin and commercially available ‘ready to use’ A23187 revealed that ionomycin is more efficient in activating both mouse and human eggs [2 followed by two treatments with ionomycin does not truly mimic a physiological Ca2+ oscillatory pattern (Caar space , whereas+ influx . In mousman eggs . However pattern .2+, mainly due to IP3-mediated Ca2+ release from intracellular stores [2+ rise induced by ethanol is longer than that observed following ionophore-mediated activation and is associated with a higher activation rate [Eggs also can be artificially activated with 7% ethanol, which results in a dramatic rise in intracellular Car stores ,242. Ethr stores ,242–244.ion rate . Ethanol2+ level results in relatively low rates of egg activation in mammals. Presumably, a transient rise in Ca2+ levels leads to inactivation of maturation promoting factor, a key Ca2+ target whose inactivation is essential for resumption of meiosis, but is not sufficient to sustain late events of egg activation that require additional Ca2+ signals [A single increase in the egg intracellular Ca signals ,245,246. signals .6.2.2.2+ signals. Therefore, chemicals that trigger Ca2+ oscillations activate eggs more efficiently than chemicals that induce a single rise, even though the eggs are flexible regarding the exact Ca2+ oscillatory pattern for activation. Strontium is the compound of choice for egg activation in rodents, but its efficacy in other species is controversial. The discovery that strontium activates mouse eggs was made inadvertently during experiments designed to examine the ability of various divalent cations to substitute for Ca2+ during sperm capacitation [2+ oscillations and supports preimplantation embryo development. Strontium-induced activation is mediated by the IP3 receptor, probably through its sensitization to Ca2+, and depends on the presence of TRPV3 in the egg [2+ transient is observed immediately after eggs are exposed to strontium, which is followed by repetitive smaller spikes resembling the oscillatory pattern observed after IVF [2+ dynamics after strontium-mediated activation remain unexplored, but it is likely that the activation failure is related to inefficient induction of Ca2+ oscillations. Strontium does not induce Ca2+ oscillations in human eggs and, consequently, fails to trigger egg activation [In mammals, the information to elicit egg activation is encoded in sperm-induced repetitive Cacitation . In the  the egg . A long fter IVF . In bovifter IVF ,250–252.tivation ,254.2+ release in several cell types. It evokes repetitive Ca2+ oscillations in eggs, as was first observed in experiments with golden hamster eggs [2+ by oxidation of sulfhydryl groups, resulting in multiple Ca2+ oscillations with similar dynamics to sperm-induced Ca2+ signalling, but egg activation is not observed with use of thimerosal alone [2+ rise that leads to egg activation in pigs [2+ signalling in the egg, it enhances Ca2+ response after IVF or other activators of Ca2+-spiking, which highlights a difference in the mechanism of Ca2+ release [2+ oscillations, whereas in sea urchin, thimerosal induces a single Ca2+ wave that closely resembles fertilization signalling [2+ oscillations.Thimerosal is a mercury-containing organic compound that induces Cater eggs . Thimeroal alone . Thimeroal alone . However in pigs . Surpris release . In cow gnalling ,259,260.6.2.3.2+ responses are likely to be more effective than those that induce non-physiological patterns. Therefore, microinjection of eggs with sperm extracts, PLCζ or IP3 to trigger egg activation is more attractive for clinical applications. Sperm extracts were first proposed for egg activation after it was shown that they had this activity. Activation rates of cloned horse embryos using stallion sperm extracts were significantly improved relative to other methods [2+ response is similar to that induced by sperm [Even though we do not fully understand the long-term effects of AEA, methods that best mimic sperm-induced Ca methods . Recentlby sperm .ζ emerged as a suitable solution. Bovine SCNT embryos activated by PLCZ1 cRNA microinjection exhibited not only a similar Ca2+ oscillatory pattern but also a similar gene expression profile when compared with IVF controls [PLCZ1 cRNA microinjection in human eggs as compared with ionomycin-mediated activation [To avoid using complex biological extracts, the use of cRNA encoding PLCcontrols . Moreovetivation . Similartivation .PLCZ1 cRNA and sperm extracts for clinical use. First, it is not possible to accurately control the expression of PLCζ protein from microinjected cRNA [ζ protein. In this regard, microinjection of human eggs with recombinant human PLCζ protein produced in E. coli overcomes clinical ICSI failure and supports embryo development to the blastocyst stage [ζ is not yet commercially available.There are several concerns regarding the use of ted cRNA . In addist stage . Despite2+ signalling after IVF. Further investigation is required to design accurate methods for egg activation that mimic physiological events, sustain embryonic development and ensure offspring health.The increasing use of artificial reproductive technologies has sparked interest among researchers and human as well as animal clinics about AEA. However, special caution needs to be taken due to the concerns regarding possible long-term effects on offspring health of abnormal Ca7.in vitro or in vivo have altered Ca2+ responses triggered by many stimuli including sperm, IP3 and PLCζ. Both the amplitude and the rate of rise of Ca2+ transients are lower in post-ovulatory aged eggs [2+ stores is smaller and that the IP3 receptor sensitivity and level of phosphorylation are decreased in aged eggs [2+ release [2+ oscillations [2+ oscillations is also shorter in post-ovulatory aged eggs [2+ oscillations [2+ influx triggers more frequent Ca2+ oscillations [2+ influx occurs during post-ovulatory ageing; this influx seems to be responsible for the lower developmental potential of aged eggs [2+ influx is altered in post-ovulatory aged eggs of other mammalian species is currently unknown. The rate of decrease of Ca2+ transients is also smaller in post-ovulatory aged eggs, probably because SERCA2 mRNA and protein levels are reduced [2+ oscillations in at least two ways [2+ uptake into the mitochondria is probably diminished. Alterations in these two processes would result in a decline in the eggs' ability to buffer cytosolic Ca2+ and could explain why the decrease in Ca2+ levels after each transient is slower in aged eggs.In mammals, after ovulation there is a defined time window for successful fertilization and development after which deterioration of the egg takes place, known as post-ovulatory ageing. Post-ovulatory aged eggs display increased fragmentation, reduced fertilization and developmental potential and, when successfully fertilized, abnormalities in the offspring . Eggs thged eggs –271. Theged eggs . Also, tged eggs . IP3 rec release . Inhibitllations . Consistged eggs ,270,273.llations ,271,273.llations . Interesged eggs . Whether reduced ,271,273.two ways ,273. ATP2+ signalling during post-ovulatory ageing. In Jurkat cells undergoing apoptosis, caspase-3 cleaves IP3R1, generating a 95 kDa C-terminal fragment that contains all transmembrane domains and the channel pore [In vitro aged eggs contain this 95 kDa fragment, which is not present in freshly ovulated eggs. Injection of mRNA encoding this fragment into fresh eggs results in fragmentation and impaired Ca2+ oscillations, suggesting that processing of the IP3 receptor is responsible, at least in part, for altered Ca2+ signalling during ageing [2+ oscillations trigger egg activation in freshly ovulated eggs, they cause apoptosis in post-ovulatory aged eggs [in vitro aged, activated egg with a freshly ovulated egg decreases fragmentation and improves developmental potential, indicating that some cytoplasmic factors are lost during ageing. One such molecule is the anti-apoptotic factor Bcl-2, whose mRNA and protein levels are reduced in aged eggs [An IP3R1 protein fragment generated by caspase-mediated cleavage may also cause altered Canel pore . In vitrg ageing . Interesged eggs . The fusged eggs .The process of post-ovulatory ageing and its detrimental effects on fertilization and embryonic development are particularly relevant for ART. An extended period of time can occur between egg retrieval and insemination, especially in ‘rescue ICSI’ cases, in which eggs that failed to fertilize following insemination are later injected with sperm. Further elucidation of the mechanisms involved in post-ovulatory ageing is key to developing protocols to alleviate this age-dependent deterioration of fertilization and developmental potential during ART procedures.8.2+ signals are modulated at fertilization. Here, we will highlight a few new areas that could have significant impacts on these Ca2+ signals but have not been studied in depth. First, altered metabolic states have broad-ranging impacts on cellular physiology through changed dynamics of reactive oxygen species. These impacts probably include Ca2+ handling, for example, by affecting the amount of Ca2+ in intracellular stores, production of ATP necessary for active Ca2+ transport or changing downstream intracellular signalling pathways. What are the impacts of altered adult metabolic status on fertilization-induced Ca2+ signals, and do these changes impact offspring health? Second, there is voluminous support in somatic cells for the idea that membrane contact sites are regions critical for Ca2+ exchange between organelles and/or the extracellular space. Furthermore, lipid-transport proteins at membrane contact sites support the rapid transport of phospholipids, perhaps including PIP2, between the ER and other membrane-bound compartments. Both of these functions suggest a role for membrane contact sites and their associated proteins in regulating Ca2+ signals at fertilization, but to date there is no such evidence. Third, to what extent do organelles besides the ER function in regulating Ca2+ signals at fertilization? There is good evidence that Ca2+ released at fertilization impacts mitochondrial ATP production [2+ regulation as well? For mammals, what is the nature of the intracellular vesicles where the PIP2 substrate for IP3 synthesis is found?Throughout the text, we pointed out areas of uncertainty or controversy regarding how Caoduction , but are2+ signalling in external fertilizers was derived from generally physiological experimental conditions, almost all that we know regarding internal fertilizers, particularly mammals, has been obtained from experiments performed in culture dishes. Two exceptions to this statement are C. elegans and Drosophila in which genetic tools, combined with the relative transparency of the relevant organs, allowed in vivo observations of Ca2+ signals at fertilization [in vitro studies reflect what occurs in vivo, but this assumption was strongly challenged by the recent findings that mouse sperm lacking PLCζ produce offspring despite highly abnormal patterns of Ca2+ signalling when studied in vitro [in vivo patterns were different from in vitro or that studies defining the necessity for a prolonged series of Ca2+ oscillations to support full-term development were only applicable in vitro. This question could be resolved with appropriate genetically encoded Ca2+ indicators combined with intravital imaging. These and other questions will keep scientists interested in understanding the Ca2+ signals responsible for the initiation of development busy for the foreseeable future.Finally, although the wealth of information we have regarding Calization ,63. We hin vitro ,92. Thes


Knocking down PMCA4 had minimal effects on numerous metabolic parameters . In summary, this study provides the first evidence that PMCA4 is over-expressed in PDAC and plays a role in cell migration and apoptotic resistance in MIA PaCa-2 cells. This suggests that PMCA4 may offer an attractive novel therapeutic target in PDAC.Pancreatic ductal adenocarcinoma (PDAC) is largely resistant to standard treatments leading to poor patient survival. The expression of plasma membrane calcium ATPase-4 (PMCA4) is reported to modulate key cancer hallmarks including cell migration, growth, and apoptotic resistance. Data-mining revealed that PMCA4 was over-expressed in pancreatic ductal adenocarcinoma (PDAC) tumors which correlated with poor patient survival. Western blot and RT-qPCR revealed that MIA PaCa-2 cells almost exclusively express PMCA4 making these a suitable cellular model of PDAC with poor patient survival. Knockdown of PMCA4 in MIA PaCa-2 cells (using siRNA) reduced cytosolic Ca Pancreatic cancer, particularly pancreatic ductal adenocarcinoma (PDAC), is a commonly diagnosed cancer with the lowest 5-year survival rate of all cancers . The ear2+) signaling has been reported to facilitate malignancies in multiple types of cancer, including PDAC i (Ca2+ overload), has been associated with apoptotic and necrotic cell death i which reaches a high steady state. Subsequent removal of external Ca2+ allows [Ca2+]i clearance, almost exclusively due to PMCA [Cells loaded with Ca to PMCA  which is2+]i clearance compared to siNT control (p = 0.0091) when compared to siNT controls (τ = 126.8 ± 22.4 s), suggesting Ca2+ clearance rate was significantly reduced as expected  = 525.8 ± 65.9, p < 0.001) compared to siNT controls  compared to siNT controls after 48 h  and siPMCA4 (37.5 ± 2.7%) compared to DMSO vehicle control , making calibration of [Ca2+]i difficult (2+]i responses was quantified by measuring the area under the curve (AUC) of uncalibrated fura-2 signal ratios  was 3.6-fold higher than siNT controls resting [Ca2+]i (79 ± 12 nM). . E These r2+ is a known modulator of both glycolytic i. Moreover, PMCA4 mediated Ca2+ signaling, in particular, has also been implicated in multiple cancer hallmarks including i can induce apoptotic cell death by Ca2+ overload, the subsequent release of cytochrome C from the mitochondria and activation of pro-apoptotic caspases. Over-expression of PMCAs in cancer prevents [Ca2+]i overload under stress stimuli and subsequently leads to apoptotic resistance i by maintaining a higher ΔΨm to avoid mitochondrial membrane depolarization which subsequently triggers apoptotic cells death i. However, loss of PMCA4 expression sensitizes PDAC cells to Ca2+ overload-associated apoptotic cell death. As PDAC is notoriously insensitive to current clinical therapies, acquiring the ability to selectively sensitize PMCA4-overexpressing PDAC cells to apoptotic-inducing chemotherapy is crucial. Therefore, targeting PMCA4 may potentially be beneficial as a therapeutic adjuvant which selectively sensitizes PDAC cells to currently available clinical therapy while sparing healthy tissues.Taken together, PMCA4 has a distinct role in Ca2 (g), at 37 °C. Mycoplasma contaminations were screened by DAPI/Hoechst 33,342 staining i) which reached a short-lived steady state due to a balance between Ca2+ entry and Ca2+ efflux. Therefore, subsequent removal of external Ca2+, by perfusion with Ca2+-free/EGTA-containing HPSS, allowed [Ca2+]i clearance to be observed and assessed. The addition of such high external Ca2+ was necessary such that [Ca2+]i clearance rate could be assessed over a much greater dynamic range of [Ca2+]i. These methods have been fully characterized in our previous studies [2+]i clearance was determined using single exponential decay fit and results expressed as a time constant (τ).  were incubated with either siNT or siPMCA4 for 48 h. Cells were loaded with 4 μM fura-2 Ca studies ,10. Fluo2+]i calibrations were calculated using the following equation: [Ca2+] nM = 541.6 nM * ((Ratio−0.47)/(2.101−Ratio)). (2+]i was quantified by the area under the curve (AUC) as previously described by James, A.D., et al. [Fura2-loaded cells were perfused with HPSS until the 340/380 nm fluorescence ratio stabilized. HPSS containing 30 μM CPA was perfused for 15 min then rinsed with CPA-free HPSS for another 15 min. 510 nm emitted light was imaged using the same method as described in 4.8. Background-subtracted images were collected at 5 sec intervals  through the MetaFluor software . All [CaRatio)).  [Ca2+]i , et al. ,10.http://imagej.net/Fiji/Downloads; Sterile 2-well culture inserts  were placed into 12 well culture plates. Cells were seeded at 50,000 cells into each side of the Ibidi insert then treated with designated siRNA for 24 h. Ibidi inserts were removed to create ‘cell-free gap’. Mitomycin C  was added to designated treatment conditions to prevent cell proliferation from confounding the cell migration results. Regions of interest (cell-free gap area) were immediately defined after Ibidi insert removal through CellSens software . Defined regions of interest were imaged at 0, 24, and 48 h. Images were collected on an Olympus IX83 inverted microscope using a 4x objective and captured using an Orca ER camera . Images were then processed, and cell-free area was quantified using Fiji ImageJ . 5 μM of caspase 3/7 green apoptosis reagent  and 625 nM Nuclear-ID Red  were added 30 minutes prior to imaging. Cells were imaged once every hour, from 0–12 hour, on the IncuCyte, using 10× lens. Background-subtracted images were analyzed using IncuCyte Zoom software and data are presented as % apoptosis [(green cell count/image ÷ red cell counts/image) × 100].Cells seeded were incubated for 48 h in either siNT or siPMCA4. Seahorse cartridges were hydrated according to manufacturer instructions. Low phenol-red DMEM, pH 7.4 media was used for all Seahorse-based assays. Either vehicle control (<0.1% DMSO) or 30 µM CPA were pre-injected in addition to the sequentially designated reagents for the mito stress  and glycolysis stress test . Seahorse data obtained were analyzed using Wave analysis software . Seahorse XF real-time ATP rate equations were used to convert basal and oligomycin treated OCR and ECAR measurements . Data are presented as means ± SEM. All data were tested for normality using the Sharpiro–Wilk test. Parametric comparisons were performed using either Student’s t-test or analysis of variance (ANOVA) with Tukey post-test. Non-parametric comparisons were performed using Mann–Whitney U-test or Kruskal–Wallis with Dunn’s multiple comparisons test. Two-factor comparisons were performed using Two-way ANOVA with Dunnett’s post-test. Statistical significance is defined as 2+ clearance, impaired PDAC cell migration and sensitized PDAC cells to Ca2+-mediated apoptosis. However, PMCA4 knockdown had minimal effects on cell viability and proliferation. This suggests that PMCA4 expression facilitates cell migration and apoptotic resistance in PDAC cells. Therefore, targeting PMCA4 over-expression in PDAC may selectively sensitize PDAC cells to apoptosis while sparing healthy tissues which express multiple PMCA isoforms.Over-expression of PMCA4 is observed in patient PDAC tumors and is correlated with poor patient survival. The current study showed that MIA PaCa-2 cells almost exclusively express PMCA4 and represent a good model to examine the role of PMCA4 in PDAC. siRNA-mediated knockdown of PMCA4 expression, which resulted in impaired Ca


DNMT3A and TP53 mutant AML by reviewing the mechanisms underlying epigenetic therapies’  resistance and providing critical points and hints for possible future therapies overcoming AML refractoriness.Acute myeloid leukemia (AML) is a clonal hematopoietic disorder characterized by abnormal proliferation, lack of cellular differentiation, and infiltration of bone marrow, peripheral blood, or other organs. Induction failure and in general resistance to chemotherapeutic agents represent a hindrance for improving survival outcomes in AML. Here, we review the latest insights in AML biology concerning refractoriness to therapies with a specific focus on cytarabine and daunorubicin which still represent milestones agents for inducing therapeutic response and disease eradication. However, failure to achieve complete remission in AML is still high especially in elderly patients (40–60% in patients >65 years old). Several lines of basic and clinical research have been employed to improve the achievement of complete remission. These lines of research include molecular targeted therapy and more recently immunotherapy. In terms of molecular targeted therapies, specific attention is given to  FLT3 mutations is an example of a subtype in which treatment with FLT3 inhibitors, although promising, is not completely efficient due to the activation of target-dependent mechanisms (acquisition of point mutations in the kinase domain) reducing enzyme-inhibitor binding or through target-independent mechanisms and leading to primary or acquired resistance. Therapeutic resistance is one of the most crucial milestones in drug treatment. Translational research has indeed focused for years on the study of this topic. A variety of biological and genetic factors has been investigated with the recent involvement of untranscribed RNA products called non-coding RNAs (ncRNAs) in the regulation of main molecular drivers of AML  ..NPM1 mutFLT3 gene mutations do not seem to be related to primary chemotherapeutic resistance while a subset of mutations in other genes such as ASXL1, SETBP1 and WT1 define specific groups of patients experiencing PIF [KMT2C, encoding a component of the myeloid/lymphoid or mixed-lineage leukemia (MLL) chromatin remodelling complex, as a possible marker of chemorefractoriness, phenocopying chromosome 7q deletion, frequently observed also in adult higher-risk myelodysplastic syndromes (MDS) and secondary AML and found to be particularly susceptible to epigenetic therapies [KMT2C (7q36.1) is shared with the EZH2 gene, the histone methyltransferase enhancer of the Zeste Homolog 2, whose mutations are observed in up to 9% of de novo and secondary AML [EZH2 mutated cell lines show a 5-fold decrease in sensitivity [The mechanisms of chemotherapeutic resistance seem to be quite variable across molecular subtypes and/or age of the patients. In pediatric patients indeed genetic mechanisms seem to be the drivers of chemoresistance. Of note is that, differently from the adult counterpart, in the pediatric population, AML cing PIF . In thisherapies ,19. The dary AML . Loss ofsitivity . In factDNMT3A, mutated in approximately 30% of NK-AML and encoding a DNA methyltransferase that catalyses 5-methylcytosine methylation, is another example of mutation affecting prognosis, being related to anthracycline resistance and PIF [DNMT3A resides in its regulatory domains which allow the interactions with histone methyltransferases. These histones modulate the expression of genes after therapies. The most common mutation in the DNMT3A gene occurs at position 882 (R882H). R882H mutation acts in a dominant-negative fashion to disrupt the de novo methyltransferase activity of normal homotetramer. Mutations in DNMT3A not only seem to co-occur with genomic alterations in FLT3 and NPM1 to induce leukemogenesis in mice but also to create DNR resistance through the blockage of DNA damage response mechanism initiated by checkpoint kinase 1 (an event necessary for DNR mechanism of action) [ and PIF . The key action) .MLL  on chromosome 11q23 is mutated in up to 10% of adult AML cases and represents another peculiar example of gene defining an AML subtype with an exquisite chemorefractoriness [FLT3 gene whose overexpression may be responsible for the poor prognosis of some MLL-rearranged AML cases, suitable for specific FLT3-inhibitors treatments [toriness . MLL-reatoriness . These ctoriness . Howevertoriness  providedtoriness /Togethertoriness ,29. A paeatments .TP53 mutations, accounting for 5–10% of de novo AML and up to 30% of therapy-related cases, represent a model of aggressive disease because of treatment failure. TP53 mutated AML patients show a characteristic genomic instability with a complex karyotype (CK) and various patterns of co-mutations according to the TP53 allelic state, making this setting particularly prone to chemoresistance [TP53 signalling disruption as a pillar in determining leukemic cell survival and aggressiveness [TP53 mutated AML and higher-risk MDS, responses are usually short-lasting and even patients achieving a morphological CR had next-generation sequencing (NGS) minimally residual disease (MRD) detectable clone(s) which later on were attributable to patients’ relapse [TP53 reads. These patients, characterized by lower levels of p21 expression, had also worse clinical outcome if compared to wild-type counterparts. In this study, the authors modelled this condition in vitro using two AML cell lines  bearing low-frequency single hotspot TP53 mutations. Resistant cells from both cell lines expressed TP53 mutations including the wild-type counterparts manifesting chemoresistance. Leukemic cells with TP53 mutations at subclonal levels had a survival advantage because of higher fractions of leukemic stem cells (LSC) (as shown in these in vitro models) which ultimately led to chemoresistance. In particular, TP53 mutation impaired GADD45A  expression in resistant cells negatively affecting Ara-C responsiveness. In the same study, the authors also showed how romidepsin, an histone deacetylase inhibitor, may restore Ara-C sensitivity via elevation of p21/GADD45A expression [Finally, sistance . Howeversiveness ,33. Alth relapse . Moreove relapse  demonstrpression .FLT3 and NPM1 mutational status may predict therapy resistance [ABCB1 gene, the multidrug resistance-associated protein 1  and the breast cancer resistance protein  [Vinca alkaloids and epipodophyllotoxins, in the attempt to defend HSCs [FLT3-ITD and not only to EZH2 mutations, possibly driving refractory to chemotherapeutic agents and depressing their sensitivity several hundred times [A large study conducted on 4601 patients with AML showed that age and performance status of the patient (host) together with other disease variables such as white blood cells (WBC) counts, disease ontogeny (primary vs. secondary), cytogenetics and sistance . Apart fsistance . Resistay ABCG2) . These Aend HSCs ,40. Diffend HSCs ,42. Studend HSCs . Overexped times ,44,45.Programa para el Tratamiento de Hemopatias Malignas) group showed no differences in terms of single nucleotide polymorphism of ABCB1 gene upon the efficacy of induction chemotherapy in AML, there was a significant association with induction death and ABCB1 triple variant haplotype [de novo AML showing the association of activating  and deactivating  genes variants with PIF [Although a recent study of the Spanish PETHEMA  signaling. As a result, FOXO proteins translocate from nucleus to cytoplasm lowering cell metabolic activities and generating an overexpression of various ABC family transporters [Apart from soluble factors, also physical characteristics of BM niches such as hypoxia or acidic pH provide a protective microenvironment for leukemic blasts . While hsporters ,65,66,67sporters ,69. In pSeveral other mechanisms of chemoresistance are not related to gene mutations or polymorphisms of detoxifying enzymes. In the last few years, new insights into cancer research paved the way for alternative mechanisms of resistance to pharmacologic agents in AML biology.Ubiquitin and SUMO proteins control a wide range of cellular functions at a posttranslational level  and theiNovel mechanisms of mediating resistance to anticancer drug activities have been introduced by the combination of microarrays/RNA-sequencing and bioinformatics. Long non-coding RNA (lncRNA) are unique RNA transcripts that play a pivotal role in cancer development and also in conferring drug resistance ,75. Havip = 0.03) and secondary AML  with also a better overall survival in the latter group  [Targeting AML metabolism has been the most recent field of research in order to optimize drug delivery systems. After the theorization of the “Combination Index”, initially proposed by Chou-Talalay in order to quantitatively outline drug antagonism, synergism and additive effects, many efforts have been made to specifically find drug formulations with the best pharmacokinetics and pharmacodynamics properties . In part = 0.01) . Nanosca = 0.01) .Furthermore, exosomes are extracellular vesicles of 30–100 nm in diameter released from body fluids with the function of delivering signaling molecules to distant cells. These vesicles serve as natural nanocarriers given the possibility to manipulate their structure for clinical use. They have been reported elevated in cancer  and reduHypomethylating agents (HMA) such as 5-azacytidine (AZA) and decitabine (DEC) represent a breakthrough in the treatment of patients with MDS and AML. However, despite their efficacy in ameliorating transfusion dependency and survival outcomes, HMA alone cannot completely cure the disease and their efficacy, even after achieving CR, sooner or later is lost if not consolidated with other approaches such as aHCT ,95. BothRecently, it has been shown that the continuous exposure to HMA, resulting in nucleotide imbalances due to off-target inhibition of thymidylate synthase and ribonucleotide reductase by DEC and AZA respectively, generates with time a weaker DNMT1-depletion . MoreoveASXL1 and EZH2 gene mutations do not rely on methylation to drive leukemia evolution and may generate subclones regardless of AZA treatment as well as some DNMT3A frameshift mutations [Resistance to AZA may be caused by genomic predisposition such as mutations, which make the clone intrinsically resistant, or epigenetic mechanisms. utations . Moreoveutations . Startinutations .Patients achieving CR and subsequently relapsing after therapy or aHCT represent another difficult clinical scenario where other mechanisms may play a major role. The genomic and transcriptomic profile of AML has clarified that AML is a disease characterized by heterogeneity of factors where molecular networks often interplay with the tumor microenvironment. Given this heterogeneity, identified by the diverse subtypes of AML in the World Health Organization classification of AML and myeloid neoplasms in general, it is conceivable that precision medicine is the frontier in AML treatment. Individualized therapies are only possible to invent or re-discover, based on the consideration that a unique therapy will not apply to all AML population or “one size does not fit all”. The current rate of failure in conventional therapies or relapses and the high mortality rates of patients failing those regimens instruct us to consider all avenues of drug-based research to reduce chemoresistance. As a proof-of-concept of the variety of actionable roads of research attenuating chemoresistance and new modalities and systems improving drug metabolisms and targeting, we created a picture in


Congenital hyperinsulinism (CHI) is a rare disease characterized by persistent hypoglycemia as a result of inappropriate insulin secretion, which can lead to irreversible neurological defects in infants. Poor efficacy and strong adverse effects of the current medications impede successful treatment. The aim of the study was to investigate new approaches to silence β-cells and thus attenuate insulin secretion.+ (KATP) channels. Additionally, KATP channel-independent targets as Ca2+-activated K+ channels of intermediate conductance (KCa3.1) and L-type Ca2+ channels were investigated. Experiments were performed using human islet cell clusters isolated from tissue of CHI patients  and islet cell clusters obtained from C57BL/6N (WT) or SUR1 knockout (SUR1-/-) mice. The cytosolic Ca2+ concentration ([Ca2+]c) was used as a parameter for the pathway regulated by electrical activity and was determined by fura-2 fluorescence. The mitochondrial membrane potential (ΔΨ) was determined by rhodamine 123 fluorescence and single channel currents were measured by the patch-clamp technique.In the scope of our research, we tested substances more selective and more potent than the gold standard diazoxide that also interact with neuroendocrine ATP-sensitive KATP channel opener NN414 (5 µM) diminished [Ca2+]c in isolated human CHI islet cell clusters and WT mouse islet cell clusters stimulated with 10 mM glucose. In islet cell clusters lacking functional KATP channels (SUR1-/-) the drug was without effect. VU0071063 (30 µM), another KATP channel opener considered to be selective, lowered [Ca2+]c in human CHI islet cell clusters. The compound was also effective in islet cell clusters from SUR1-/- mice, showing that [Ca2+]c is influenced by additional effects besides KATP channels. Contrasting to NN414, the drug depolarized ΔΨ in murine islet cell clusters pointing to severe interference with mitochondrial metabolism. An opener of KCa3.1 channels, DCEBIO (100 µM), significantly decreased [Ca2+]c in SUR1-/- and human CHI islet cell clusters. To target L-type Ca2+ channels we tested two already approved drugs, dextromethorphan (DXM) and simvastatin. DXM (100 µM) efficiently diminished [Ca2+]c in stimulated human CHI islet cell clusters as well as in stimulated SUR1-/- islet cell clusters. Similar effects on [Ca2+]c were observed in experiments with simvastatin (7.2 µM).The selective KATP channel opener diazoxide. Targeting KCa3.1 channels by channel openers or L-type Ca2+ channels by DXM or simvastatin might be valuable approaches for treatment of CHI caused by mutations of KATP channels not sensitive to KATP channel openers.NN414 seems to provide a good alternative to the currently used K ABCC8 and KCNJ11. ABCC8 and KCNJ11 genes encode the KATP channel subunits SUR1 and Kir6.2, respectively, and mutations in these genes represent the most prevalent cause of CHI. Defects in these genes are responsible for the failure of β-cells to respond to normal regulatory mechanisms, leading to inappropriate and excessive insulin release despite low blood glucose concentrations resulting in frequent episodes of hypoglycemia  is a rare heterogeneous genetic disorder, but the most frequent cause of severe, persistent hypoglycemia in neonates, infants and children. The main reasons for developing CHI are defects in important genes regulating pancreatic β-cell function. To date, mutations in 14 essential genes controlling insulin secretion have been reported including glycemia .Based on histopathological observations, three distinct forms of CHI are described: focal, diffuse and atypical. In focal CHI affected β-cells are localized only in small specific parts of the pancreas. Conversely, in diffuse CHI all pancreatic β-cells seem to be affected . If the ATP channel agonist diazoxide . Written informed consent was provided by the legal guardians of the children for the study. The islets were taken from biopsies of eight CHI patients. Genetic studies showed that seven patients had mutations in the -/-) mice on a C57BL/6N background. The mice were bred in the animal facility of the Department of Pharmacology at the University of Tübingen. The principles of laboratory animal care  and German laws were followed. The animal study was reviewed and approved by the Regierungspräsidium Tübingen (§ 4 Abs. 3 TierSchG). Islets were isolated and cultured as previously described (Mouse islets of Langerhans were isolated from adult C57BL/6N (WT) mice or SUR1 knockout : 140 NaCl, 5 KCl, 1.2 MgCl2, 2.5 CaCl2, 10 HEPES and glucose as indicated, pH 7.4 adjusted with NaOH. The same bath solution was used for the determination of the mitochondrial membrane potential (ΔΨ). The pipette solution for single channel recording contained (in mM): 130 KCl, 1.2 MgCl2, 2 CaCl2, 10 EGTA, and 10 HEPES; pH was adjusted to 7.4 with KOH. The bath solution contained (in mM): 130 KCl, 2 CaCl2, 10 EGTA, 1 Na2ATP, 1.7 MgCl2, and 20 HEPES with pH adjusted to 7.2 with KOH.Measurements of c under different experimental conditions.Details are described in . In brievice versa.ΔΨ was measured by Rh123 fluorescence at 480 nm excitation wavelength as described in . One datE1507K protein together with WT human cDNA for Kir6.2 was expressed in a stably transfected HEK-293 cell line ; inward currents are shown as downward deflections. Patch pipettes had a resistance of 6–8 MΩ. Currents were recorded with an EPC-9 patch-clamp amplifier using Patchmaster software . Analyses to estimate mean current were done offline in IgorPro 7 . With diazoxide or NN414 so many channels open simultaneously that a single channel evaluation of open probability (Po) was not possible. We therefore evaluated the mean current for 20 s before the end of a maneuver.Patch-clamp recordings were done in the inside-out configuration. Kt test. Multiple comparisons were made by ANOVA followed by Student-Newman-Keuls test. P values ≤ 0.05 were considered significant.Each series of experiments with islet cell clusters from mice was performed with at least three independent mouse preparations. The number of preparations for recordings with human islet cell clusters varied and is indicated for every series. Box plots were generated using Graphpad Prism 8. Boxes correspond to the interquartile range, the line within the box to the median, and the cross to the mean. Whiskers correspond to the maximum and minimum values. Statistical significance of differences was assessed by Student’s 2+ concentration ([Ca2+]c) are driven by fluctuations of the membrane potential and [Ca2+]c is the trigger signal for insulin secretion. Consequently, [Ca2+]c is a very robust surrogate parameter for insulin secretion. It can be determined easily and online with few cell material, which is an enormous advantage when working with human tissue. Glucose-stimulated insulin secretion in human pancreatic β-cells is completely suppressed by pharmacologic blockage of L-type Ca2+ channels (2+ channel blocker nifedipine on [Ca2+]c in human islet cell clusters isolated from tissue of a patient with diffuse CHI (2+]c on top of a plateau in the presence of a stimulating glucose concentration of 10 mM. The addition of nifedipine at a concentration of 5 µM diminished [Ca2+]c significantly (Oscillations of the cytosolic Cachannels . This meATP channels (SUR1/Kir6.2) of pancreatic β-cells play a crucial role as they couple cellular metabolism to electrical activity. In electrically inactive β-cells, KATP channels are open. CHI is characterized by permanently active β-cells and thus opening of these channels is one strategy to treat it. Diazoxide is an opener of KATP channels that is already established in CHI therapy. We tested the effect of diazoxide on [Ca2+]c as a control. In human islet cell clusters isolated from two patients, one with focal and one with mosaic form of CHI   . As expeATP channel openers affect mitochondrial function in addition to their direct influence on KATP channels (-/- islet cell cluster. The switch from 0.5 to 10 mM glucose is accompanied by a strong decrease in Rh123 fluorescence reflecting hyperpolarization of ΔΨ and thus ATP production upon the increase of glucose concentration (-/- mice NN414 (5 µM) exerted any effect on ΔΨ . Addition of 1 mM ATP rapidly inhibited channel activity as expected. Application of diazoxide (340 µM) or NN414 (5 µM) in the presence of ATP led to opening of SUR1E1507K/WT Kir 6.2 channels.  to islet cells clusters from pancreatic tissue with a focal or diffuse lesion  suppressed Ca2+ oscillations and lowered [Ca2+]c in both genotypes   play an important role in the K+ current (Kslow) that contributes to β-cell hyperpolarization at the end of a burst phase with electrical activity  . Dependion (KCa) –76. It hactivity , 77, 78.activity  demonstr current , the sulCa3.1 opener DCEBIO on islet cell clusters isolated from SUR1-/- mice. DCEBIO (100 µM) effectively abolished the glucose-induced oscillations of [Ca2+]c  is a known antagonist of NMDA receptors. Active NMDA receptors can activate other ion channels, like Ca outflow . A block outflow . Lesser-trations . Since p2+]c of a permanently depolarized islet cell cluster from a SUR1-/- mouse in the presence of 10 mM glucose and application of 100 µM DXM. The drug significantly lowered the mean fluorescence ratio , e.g., in , a mutation affecting splicing (patient 3) and two nonsense mutations . The missense and splicing mutations studied possibly allow production of SUR1 proteins albeit at reduced function or level. For nonsense mutation c.3970G>T p.(Glu1324*) detected in exon 32 of patient 4, nonsense-mediated RNA decay (NMD) has been predicted in silico, however, clinically the patient was reported to be diazoxide-responsive at dosage 8mg/kg/d. This may suggest escape of NMD with this particular mutation and generation of a truncated SUR1 protein lacking the last encoded six exons but retaining residual channel function. For one patient (patient 2) no mutation was found in the KATP channel genes (ABCC8 and KCNJ11) or in other CHI genes tested. This is not unusual in clinical routine. As this patient displayed a partial response to diazoxide (see ATP (SUR1/Kir6.2) channels  and exerts weak stimulatory effects on KATP channels of the cardiac muscle (SUR2A/Kir6.2)  . One of /Kir6.2) , 92.ATP channel openers more specific for β-cells. In comparison to diazoxide, NN414 is reported to be a selective SUR1 agonist, 100-fold more potent than diazoxide, suggesting that the drug is effective at much lower concentrations  The removal of VU0071063 was followed by a transient drop in [Ca2+]c in WT and SUR1-/- islet cell clusters, which is presumably due to ATP-dependent SERCA activation. VU0071063 rapidly and significantly decreased [Ca2+]c in SUR1-/- islet cell clusters, too. This seems paradoxical as the ATP depletion leads to Ca2+ release out of the Ca2+ stores; however, the decreased [Ca2+]c during application of the drug might be secondary to Ca2+-dependent inactivation of L-type Ca2+ channels. Our data suggest that the Ca2+-lowering effect of VU0071063 is caused by a dual mechanism: 1) direct opening of KATP channels and indirect opening of KATP channels by ATP depletion; 2) interference with SERCA function and Ca2+ release, thereby mediating unpredictable interactions with other ion channels. In conclusion, since VU0071063 raises the expectation of detrimental effects on mitochondria, thereby impairing all ATP-dependent processes, this compound seems not to be suitable for use in humans without structural modifications avoiding this side effect. Noteworthy, NN414 did not affect ΔΨ , sirolimus and exendin-(9–39) (+ (GIRK) channels, decrease in Ca2+ influx via P/Q-type Ca2+ channels and inhibition of exocytosis . Ocn-(9–39) . Activatocytosis , 108. Inocytosis , 110. Hoocytosis . Sirolimocytosis , 112. Itocytosis . It has ocytosis . Furtherocytosis . HoweverATP channels, Ca2+-activated K+ channels of intermediate conductance  contribute to Kslow, the hyperpolarizing current that terminates bursts of action potentials in β-cells   –117.2+ channels than to its known target, the NMDA receptor . Written informed consent was provided by the legal guardians of the children for the study. The animal study was reviewed and approved by the Ethics Committee of Regierungspraesidium Tuebingen.JS researched the data and wrote and edited the manuscript. TH, AG, and JK researched the data, contributed to the discussion, and edited the manuscript. WB, CW, and UL provided human β-cells and contributed to discussion. IW supported the manuscript as an expert in CHI genetics and contributed to the discussion. MD and PK-D contributed to discussion and study design and edited the manuscript. GD designed the study, wrote and edited the manuscript, and contributed to discussion. GD is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors contributed to the article and approved the submitted version.This work was supported by grants from the DFG (DR225/11-1 to GD and INST 211/647-1 FUGG to MD).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.