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

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

The eggstraordinary story of how life begins

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

The role of Ca2+ in oocyte activation during In Vitro fertilization: Insights into potential therapies for rescuing failed fertilization

At fertilization the mature mammalian oocyte is activated to begin development by a sperm-induced series of increases in the cytosolic free Ca²⁺ concentration. These so called Ca²⁺ oscillations, or repetitive Ca²⁺ spikes, are also seen after intracytoplasmic sperm injection (ICSI) and are primarily triggered by a sperm protein called phospholipase Czeta (PLCζ). Whilst ICSI is generally an effective way to fertilizing human oocytes, there are cases where oocyte activation fails to occur after sperm injection. Many such cases appear to be associated with a PLCζ deficiency. Some IVF clinics are now attempting to rescue such cases of failed fertilization by using artificial means of oocyte activation such as the application of Ca²⁺ ionophores. This review presents the scientific background for these therapies and also considers ways to improve artificial oocyte activation after failed fertilization.     

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

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

Sperm PLCζ: From structure to Ca oscillations,egg activation and therapeutic potential

Significant evidence now supports the assertion that cytosolic calcium oscillations during fertilization in mammalian eggs are mediated by a testis-specific phospholipase C (PLC), termed PLC-zeta (PLCζ) that is released into the egg following gamete fusion. Herein, we describe the current paradigm of PLCζ in this fundamental biological process, summarizing recent important advances in our knowledge of the biochemical and physiological properties of this enzyme. We describe the data suggesting that PLCζ has distinct features amongst PLCs enabling the hydrolysis of its substrate, phosphatidylinositol 4,5-bisphosphate (PIP₂) at low Ca²⁺ levels. PLCζ appears to be unique in its ability to target PIP₂ that is present on intracellular vesicles. We also discuss evidence that PLCζ may be a significant factor in human fertility with potential therapeutic capacity.     

Initiation of the activation potential by an increase in intracellular calcium in eggs of the frog,Rana pipiens

The membrane potential of the frog egg undergoes a transient positive shift at fertilization which is a block to polyspermy. This paper addresses the question of how a sperm elicits this “fertilization potential.” Iontophoretic injection of Ca²⁺ activates Rana pipiens eggs to develop and initiates a transient, positive-going shift in the membrane potential (the activation potential) which is like the sperm-induced fertilization potential in amplitude, duration, and Cl^? dependence. Activation potentials are elicited by Ca² injection into both animal and vegetal regions of the egg, but the rate of the initial depolarization is much less when Ca²⁺ is injected into the vegetal region. Injections of K⁺, Na⁺, Cl^?, or Mg²⁺ do not result in activation potentials, but the Ca²⁺ analogs, Sr²⁺ and Ba²⁺, can substitute for Ca²⁺. Treatment of eggs with the divalent cation ionophore, A23187, also initiates a transient, positive-going depolarization. Because injection of Ca²⁺ is sufficient to elicit a response almost identical to a fertilization potential, the ion transport mechanisms necessary to produce a fertilization potential must preexist in the unfertilized eggs; the sperm contributes only the stimulus to activate these mechanisms. The results reported here suggest that the stimulus may be a rise in free Ca²⁺.     

Identification of phospholipase C zeta in normospermic and teratospermic domestic cat sperm

In mammalian species, oocyte activation is initiated by oscillations in the intracellular concentration of free calcium ([Ca²⁺]_i), which are also essential to allow embryonic development. To date, evidence supporting the hypothesis that a sperm factor is responsible for initiating oocyte activation has been presented in various mammalian species. Among the possible candidates to be the active sperm factor is the novel sperm-specific phospholipase C ζ (PLCζ), which besides its testis-specific expression is capable of initiating [Ca²⁺]_i oscillations. In this study, we investigated the presence of PLCζ in the sperm of the domestic cat and whether normospermic and teratospermic cats differ in their PLCζ expression. Immunoblotting with anti-PLCζ antibodies confirmed the presence of an immunoreactive band of ∼70 kDa in whole sperm lysates of domestic cat as well as in both soluble and “insoluble” fractions from this sperm. Additional immunoreactive bands, probably C- and N-terminal truncated versions of PLCζ, were also visualized in the soluble sperm fractions. Interestingly, immunoreactivity of PLCζ was detectable in teratospermic sperm, although with slightly less intensity than in normospermic sperm. In conclusion, domestic cat sperm express PLCζ in both cytosolic and high-pH fractions, which is consistent with data in other mammals. Sperm from teratospermic cats also express PLCζ, albeit at reduced concentrations, which may affect the fertility of these males.     

Activation of Barnea candida (mollusca,pelecypoda) oocytes by sperm or KCl,but not by NH4Cl,requires a calcium influx

Artificial activation of Barnea candida oocytes by NH₄Cl and the addition of excess KCl are, respectively, independent and dependent upon external Ca²⁺ concentration. The activating efficiency of NH₄Cl increases when external pH is raised which suggests that the proportion of unionized penetrating NH₃ is the key factor for activation under these conditions. The external Ca²⁺-dependent period for KCl or sperm-induced activation does not exceed 3–4 min. The transmembrane Ca²⁺ flux inhibitor D-600 reversibly inhibits KCl- or sperm-induced activation and the D-600-sensitive period closely corresponds to the Ca²⁺-dependent period. Ca²⁺ ions alone can also trigger activation provided external Mg²⁺ concentration is lowered. Finally, a direct demonstration is presented for the existence of a D-600-sensitive Ca²⁺ uptake following KCl- or sperm-induced activation, by using the radioactive tracer ⁴⁵Ca. The significance of that Ca²⁺ influx is discussed with respect to the possible involvement of an intracellular pH shift as a main factor in the activation of lamellibranch mollusc oocytes.     

Mouse strain‐dependent egg factors regulate calcium signals at fertilization

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

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

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

Effects of Calcium-BAPTA Buffers and the Calmodulin Antagonist W-7 on Mouse Egg Activation

Results of numerous experiments indicate that the transient rise in intracellular Ca²⁺following sperm–egg fusion is essential for the subsequent events that constitute egg activation. Some events of egg activation, e.g., cortical granule exocytosis, however, appear more sensitive to intracellular Ca²⁺than other events, e.g., cell cycle resumption. To examine if specific events of egg activation have different thresholds for Ca²⁺, we manipulated buffered intracellular Ca²⁺concentrations by microinjecting Ca²⁺-BAPTA buffers and then examined the effect on the cortical granule exocytosis, recruitment of maternal mRNAs, and cell cycle resumption. We find that whereas cortical granule exocytosis occurs over a narrow threshold range of injected free Ca²⁺concentrations between 0.5 and 1.0 μM,recruitment of maternal mRNAs is only partially stimulated at injected free Ca²⁺concentrations of 2.5 μM,and no evidence for cell cycle resumption was observed (up to 2.5 μMCa²⁺). Although the Ca²⁺- and phospholipid-dependent protein kinase, protein kinase C, is implicated in aspects of egg activation, calmodulin is also a potential target for the transient increase in Ca²⁺that occurs following fertilization. Whereas incubation of eggs in the presence of the calmodulin antagonist W-7 followed by insemination does not block cortical granule exocytosis, cell cycle resumption, as assessed by the metaphase-to-anaphase transition, a decrease in histone H1 kinase activity and the time course for the emission of the second polar body are significantly delayed/inhibited.     

The Ca increase by the sperm factor in physiologically polyspermic newt fertilization: Its signaling mechanism in egg cytoplasm and the species-specificity

The newt, Cynops pyrrhogaster, exhibits physiological polyspermic fertilization, in which several sperm enter an egg before egg activation. An intracellular Ca²⁺ increase occurs as a Ca²⁺ wave at each sperm entry site in the polyspermic egg. Some Ca²⁺ waves are preceded by a transient spike-like Ca²⁺ increase, probably caused by a tryptic protease in the sperm acrosome at the contact of sperm on the egg surface. The following Ca²⁺ wave was induced by a sperm factor derived from sperm cytoplasm after sperm–egg membrane fusion. The Ca²⁺ increase in the isolated, cell-free cytoplasm indicates that the endoplasmic reticulum is the major Ca²⁺ store for the Ca²⁺ wave. We previously demonstrated that citrate synthase in the sperm cytoplasm is a major sperm factor for egg activation in newt fertilization. In the present study, we found that the activation by the sperm factor as well as by fertilizing sperm was prevented by an inhibitor of citrate synthase, palmitoyl CoA, and that an injection of acetyl-CoA or oxaloacetate caused egg activation, indicating that the citrate synthase activity is necessary for egg activation at fertilization. In the frog, Xenopus laevis, which exhibits monospermic fertilization, we were unable to activate the eggs with either the homologous sperm extract or the Cynops sperm extract, indicating that Xenopus sperm lack the sperm factor for egg activation and that their eggs are insensitive to the newt sperm factor. The mechanism of egg activation in the monospermy of frog eggs is quite different from that in the physiological polyspermy of newt eggs.     

Phospholipase C-η1 is activated by intracellular Ca(2+) mobilization and enhances GPCRs/PLC/Ca(2+) signaling

Phospholipase C-η1 (PLC-η1) is the most recently identified PLC isotype and is primarily expressed in nerve tissue. However, its functional role is unclear. In the present study, we report for the first time that PLC-η1 acts as a signal amplifier in G protein-coupled receptor (GPCR)-mediated PLC and Ca²⁺ signaling. Short-hairpin RNA (shRNA)-mediated knockdown of endogenous PLC-η1 reduced lysophosphatidic acid (LPA)-, bradykinin (BK)-, and PACAP-induced PLC activity in mouse neuroblastoma Neuro2A (N2A) cells, indicating that PLC-η1 participates in GPCR-mediated PLC activation. Interestingly, ionomycin-induced PLC activity was significantly decreased by PLC-η1, but not PLC-η2, knockdown. In addition, we found that intracellular Ca²⁺ source is enough for PLC-η1 activation. Furthermore, the IP₃ receptor inhibitor, 2-APB, inhibited LPA-induced PLC activity in control N2A cells, whereas this effect was not observed in PLC-η1 knockdown N2A cells, suggesting a pivotal role of intracellular Ca²⁺ mobilization in PLC-η1 activation. Finally, we found that LPA-induced ERK1/2 phosphorylation and expression of the downstream target gene, krox-24, were significantly decreased by PLC-η1 knockdown, and these knockdown effects were abolished by 2-APB. Taken together, our results strongly suggest that PLC-η1 is activated via intracellular Ca²⁺ mobilization from the ER, and therefore amplifies GPCR-mediated signaling.     

Linoleic Acid Stimulates [Ca2+]i Increase in Rat Pancreatic Beta-Cells through Both Membrane Receptor- and Intracellular Metabolite-Mediated Pathways

The role of the free fatty acid (FFA) receptor and the intracellular metabolites of linoleic acid (LA) in LA-stimulated increase in cytosolic free calcium concentration ([Ca²⁺]i) was investigated. [Ca²⁺]i was measured using Fura-2 as indicator in rat pancreatic β-cells in primary culture. LA (20 µM for 2 min) stimulated a transient peak increase followed by a minor plateau increase in [Ca²⁺]i. Elongation of LA stimulation up to 10 min induced a strong and long-lasting elevation in [Ca²⁺]i. Activation of FFA receptors by the non-metabolic agonist GW9508 (40 µM for 10 min) resulted in an increase in [Ca²⁺]i similar to that of 2-min LA treatment. Inhibition of acyl-CoA synthetases by Triacsin C suppressed the strong and long-lasting increase in [Ca²⁺]i. The increase in [Ca²⁺]i induced by 2 min LA or GW9508 were fully eliminated by exhaustion of endoplasmic reticulum (ER) Ca²⁺ stores or by inhibition of phospholipase C (PLC). Removal of extracellular Ca²⁺ did not influence the transient peak increase in [Ca²⁺]i stimulated by 2 min LA or GW9508. The strong and long-lasting increase in [Ca²⁺]i induced by 10 min LA was only partially suppressed by extracellular Ca²⁺ removal or thapsigargin pretreatment, whereas remaining elevation in [Ca²⁺]i was eliminated after exhaustion of mitochondrial Ca²⁺ using triphenyltin. In conclusion, LA stimulates Ca²⁺ release from ER through activation of the FFA receptor coupled to PLC and mobilizes mitochondrial Ca²⁺ by intracellular metabolites in β-cells.     

Inhibitory effects of flavonoids isolated from Fragaria ananassa Duch on IgE-mediated degranulation in rat basophilic leukemia RBL-2H3

We isolated the 4 kinds of flavonoids from strawberry ‘Nohime’ and examined the effect of these flavonoids on the degranulation in RBL-2H3 cells. The flavonoids were found to suppress the degranulation from Ag-stimulated RBL-2H3 cells to different extents. To disclose the inhibitory mechanism of degranulation by flavonoids, we examined their effects on the intracellular free Ca²⁺ concentration ([Ca²⁺]i) and the intracellular signaling pathway such as Lyn, Syk, and PLCγs. The intracellular free Ca²⁺ concentration ([Ca²⁺]i) was elevated by FcεRI activation, but these flavonoid treatments reduced the elevation of [Ca²⁺]i by suppressing Ca²⁺ influx. Kaempferol strongly suppressed the activation of Syk and PLCγs. It was thus suggested that suppression of Ag-stimulated degranulation by the flavonoids is mainly due to suppression of [Ca²⁺]i elevation and Syk activation. These results suggested that strawberry would be of some ameliorative benefit for the allergic symptoms.     

Imaging Calcium in Drosophila at Egg Activation

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. One aspect of egg activation, conserved across all organisms examined, is a change in the intracellular concentration of calcium (Ca²⁺) often termed a ''Ca²⁺ wave''. While the speed and number of oscillations of the Ca²⁺ wave varies between species, the change in intracellular Ca²⁺ is key in bringing about essential events for embryonic development. These changes include resumption of the cell cycle, mRNA regulation, cortical granule exocytosis, and rearrangement of the cytoskeleton.In the mature Drosophila egg, activation occurs in the female oviduct prior to fertilization, initiating a series of Ca²⁺-dependent events. Here we present a protocol for imaging the Ca²⁺ wave in Drosophila. This approach provides a manipulable model system to interrogate the mechanism of the Ca²⁺ wave and the downstream changes associated with it.     

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

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

The extracellular calcium‐sensing receptor promotes porcine egg activation via calcium/calmodulin‐dependent protein kinase II

Extracellular calcium is required for intracellular Ca²⁺ oscillations needed for egg activation, but the regulatory mechanism is still poorly understood. The present study was designed to demonstrate the function of calcium‐sensing receptor (CASR), which could recognize extracellular calcium as first messenger, during porcine egg activation. CASR expression was markedly upregulated following egg activation. Functionally, the addition of CASR agonist NPS R‐568 significantly enhanced pronuclear formation rate, while supplementation of CASR antagonist NPS2390 compromised egg activation. There was no change in NPS R‐568 group compared with control group when the egg activation was performed without extracellular calcium addition. The addition of NPS2390 precluded the activation‐dependent [Ca²⁺]_i rise. When egg activation was conducted in intracellular Ca²⁺ chelator BAPTA‐AM and NPS R‐568 containing medium, CASR function was abolished. Meanwhile, CASR activation increased the level of the [Ca²⁺]_i effector p‐CAMKII, and the presence of KN‐93, an inhibitor of CAMKII, significantly reduced the CASR‐mediated increasement of pronuclear formation rate. Furthermore, the increase of CASR expression following activation was reversed by inhibiting CAMKII activity, supporting a positive feedback loop between CAMKII and CASR. Altogether, these findings provide a new pathway of egg activation about CASR, as the extracellular Ca²⁺ effector, promotes egg activation via its downstream effector and upstream regulator CAMKII.     

An incretin-based tri-agonist promotes superior insulin secretion from murine pancreatic islets via PLC activation

Recently, a unimolecular tri-agonist with activity at glucagon-like peptide 1 receptor (GLP-1R), glucose dependent insulinotropic receptor, and the glucagon receptor was reported to improve glycemic control in mice. Here, we defined the underlying molecular mechanisms of enhanced insulin secretion in murine pancreatic islets for a specific tri-agonist. The tri-agonist induced an increase in insulin secretion from murine islets compared to the respective mono-agonists. GLP-1R mainly signals via activation of the Gα_s pathway, but inhibition of protein kinase A (H89) and exchange protein activation by cAMP (EPAC) (ESI-09) could not completely block insulin release induced by tri-agonist. Electrophysiological observations identified a strong increase of intracellular Ca²⁺ concentration and whole-cell currents induced by tri-agonist via transient receptor potential channels (TRPs). Although, EPAC activation mobilizes intracellular Ca²⁺ via TRPs, the TRPs blockers (La³⁺ and Ruthenium Red) had a larger inhibitory impact than ESI-09 on tri-agonist stimulatory effects. To test for other potential mechanisms, we blocked PLC activity (U73122) which reduced the superior effect of tri-agonist to induce insulin secretion, and partially inhibited the induced Ca²⁺ influx. This result suggests that the relative effect of tri-agonist on insulin secretion caused by GLP-1R agonism is mediated mainly via Gα_s signaling and partially by activation of PLC. Therefore, the large portion of the increased intracellular Ca²⁺ concentration and the enhanced whole-cell currents induced by tri-agonist might be attributable to TRP channel activation resulting from signaling through multiple G-proteins. Here, we suggest that broadened intracellular signaling may account for the superior in vivo effects observed with tri-agonism.