Activation of sea urchin eggs by inositol phosphates is independent of external calcium.

We investigated the contribution of external calcium ions to inositol phosphate-induced exocytosis in sea urchin eggs. We show that: (a) inositol phosphates activate eggs of the sea urchin species Lytechinus pictus and Lytechinus variegatus independently of external calcium ions; (b) the magnitude and duration of the inositol phosphate induced calcium changes are independent of external calcium; (c) in calcium-free seawater, increasing the volume of inositol trisphosphate solution injected decreased the extent of egg activation; (d) eggs in calcium-free sea water are more easily damaged by microinjection; microinjection of larger volumes increased leakage from eggs pre-loaded with fluorescent dye. We conclude that inositol phosphates do not require external calcium ions to activate sea urchin eggs. This is entirely consistent with their role as internal messengers at fertilization. The increased damage caused to eggs in calcium-free seawater injected with large volumes may allow the EGTA present in the seawater to enter the egg and chelate any calcium released by the inositol phosphates. This may explain the discrepancy between this and earlier reports.     

Activators of protein kinase C trigger cortical granule exocytosis, cortical contraction, and cleavage furrow formation in Xenopus laevis oocytes and eggs

Prophase I oocytes, free of follicle cells, and metaphase II eggs of the amphibian Xenopus laevis were subjected to transient treatments with the protein kinase C activators, phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-didecanoate, and 1-olyeoyl-2-acetyl-sn-glycerol. In both oocytes and eggs, these treatments triggered early events of amphibian development: cortical granule exocytosis, cortical contraction, and cleavage furrow formation. Surprisingly, activation of oocytes occurred in the absence of meiotic resumption, resulting in cells with an oocytelike nucleus and interior cytoplasm, but with a zygotelike cortex. PMA-induced activation of oocytes and eggs did not require external calcium, a prerequisite for normal activation of eggs. PMA-induced activation of eggs was inhibited by retinoic acid, a known inhibitor of protein kinase C. In addition, pretreatment of eggs with retinoic acid prevented activation by mechanical stimulation and inhibited activation by calcium ionophore A23187. The results suggest that protein kinase C activation is an integral component of the Xenopus fertilization pathway.     

A wave of free cytosolic calcium traverses zebrafish eggs on activation.

The activation process in a variety of deuterostome and protostome eggs is accompanied by cytosolic calcium transients that usually take the form of either a single or multiple propagating waves. Here we report that the eggs of zebrafish (Danio rerio) are no exception in that they generate a single activation wave that traverses the egg at a velocity of around 9 microm/s. There appears, however, to be no difference between the calcium-mediated activation response of eggs with regard to the presence or absence of sperm in the spawning medium. This leads us to suggest that these eggs are normally activated when they come in contact with their spawning medium and are then subsequently fertilized. The aspermic wave is initiated at the animal pole in the region of the micropyle, appears to propagate mainly through the yolk-free egg cortex, and then terminates at the vegetal pole. As neither sperm nor external calcium is required for the initiation (or propagation) of the activation wave, this suggests that an alternative wave trigger must be involved.     

The activation wave of calcium in the ascidian egg and its role in ooplasmic segregation

We have studied egg activation and ooplasmic segregation in the ascidian Phallusia mammillata using an imaging system that let us simultaneously monitor egg morphology and calcium-dependent aequorin luminescence. After insemination, a wave of highly elevated free calcium crosses the egg with a peak velocity of 8-9 microns/s. A similar wave is seen in egg fertilized in the absence of external calcium. Artificial activation via incubation with WGA also results in a calcium wave, albeit with different temporal and spatial characteristics than in sperm-activated eggs. In eggs in which movement of the sperm nucleus after entry is blocked with cytochalasin D, the sperm aster is formed at the site where the calcium wave had previously started. This indicates that the calcium wave starts where the sperm enters. In 70% of the eggs, the calcium wave starts in the animal hemisphere, which confirms previous observations that there is a preference for sperm to enter this part of the egg (Speksnijder, J. E., L. F. Jaffe, and C. Sardet. 1989. Dev. Biol. 133:180-184). About 30-40 s after the calcium wave starts, a slower (1.4 microns/s) wave of cortical contraction starts near the animal pole. It carries the subcortical cytoplasm to a contraction pole, which forms away from the side of sperm entry and up to 50 degrees away from the vegetal pole. We propose that the point of sperm entry may affect the direction of ooplasmic segregation by causing it to tilt away from the vegetal pole, presumably via some action of the calcium wave.     

Role of calcium influx during the latent period in sea urchin fertilization

After ∼7–40 s following gamete fusion, a steadily increasing fraction of a sea urchin's zygotes initiate an activating calcium wave. The fertilization membrane then rises, the cell cycle resumes and development begins. This study focuses on the so-called latent period that occurs between the time that gamete fusion occurs and the initiation of the activating calcium wave. We inhibited calcium influx during this period by adding lanthanum or by reducing external calcium with a buffer at various time points after insemination. Both of these treatments blocked the activation of eggs that had not yet started a wave at the time of treatment. This indicates that an influx of calcium is needed during the latent period to induce egg activation. These results support the sperm conduit model of egg activation in the sea urchin, where calcium flows from the sea through the fused sperms' acrosomal process into a cortical region of the eggs' endoplasmic reticulum.     

External calcium concentration and fertilization in the sea urchin, Strongylocentrotusfranciscansus (A. Agassiz) (Echinodermata)

The role of external calcium ions in the fertilization of the sea urchin, Strongylocentrotusfranciscansus (A. Agassiz), was studied. When eggs were inseminated with jelly-treated sperm in artificial sea waters containing various concentrations of calcium, the percentage of fertilization decreased with decreasing concentrations of calcium. A small amount of external calcium ions was also found to be essential for fertilization by spermatozoa with reacted acrosomes. The binding capacity of jelly-treated spermatozoa to eggs was, however, not adversely affected by a calcium deficiency. These results suggest that calcium ions are required not only for the initiation of the acrosome reaction, but also for successful levels of fertilization following sperm-binding to eggs even after the acrosome reaction has occurred.     

External calcium ions are requisite for fertilization of sea urchin eggs by spermatozoa with reacted acrosomes

That a small amount of external calcium ions is requisite for the fertilization by spermatozoa with reacted acrosomes was found by some simple experiments using jelly-treated sperm of the sea urchin, Hemicentrotus pulcherrimus. When eggs were inseminated with the jelly-treated sperm in artificial seawaters containing calcium at various concentrations, the percentage of fertilization decreased concomitant with the reduction in the amount of external calcium ions, 50% at 40 μM calcium and almost 0% at less than 10 μM. On the other hand, it was observed that both the morphology of the reacted acrosome and the binding capacity of the jelly-treated spermatozoa to eggs were not influenced by the calcium deficiency. These results suggest that external calcium ions are indispensable even for the fertilization processes following sperm binding to eggs after the acrosome reaction, such as penetration of reacted spermatozoa through vitelline layer and/or membrane fusion between egg and spermatozoon.     

Roles of calcium and pH in activation of eggs of the medaka fish, Oryzias latipes

Unfertilized eggs of the medaka fish (Oryzias latipes) were injected with pH-buffered calcium buffers. Medaka egg activation is accompanied by a transient increase in cytoplasmic free calcium (Gilkey, J. C., L. F. Jaffe, E. B. Ridgway, and G. T. Reynolds, 1978, J. Cell Biol., 76:448-466). The calcium buffer injections demonstrated that (a) the threshold free calcium required to elicit the calcium transient and activate the egg is between 1.7 and 5.1 microM at pH 7.0, well below the 30 microM reached during the transient, and (b) buffers which hold free calcium below threshold prevent activation of the buffered region in subsequently fertilized eggs. Therefore an increase in free calcium is necessary and sufficient to elicit the calcium transient, and the calcium transient is necessary to activate the egg. Further, these results are additional proof that the calcium transient is initiated and propagated through the cytoplasm by a mechanism of calcium- stimulated calcium release. Finally, a normal calcium transient must propagate through the entire cytoplasm to ensure normal development. Unfertilized eggs were injected with pH buffers to produce short-term, localized changes in cytoplasmic pH. The eggs were then fertilized at various times after injection. In other experiments, unfertilized and fertilized eggs were exposed to media containing either NH4Cl or CO2 to produce longer term, global changes in cytoplasmic pH. These treatments neither activated the eggs nor interfered with the normal development of fertilized eggs, suggesting that even if a natural change in cytoplasmic pH is induced by activation, it has no role in medaka egg development. The injected pH buffers altered the rate of propagation of the calcium transient through the cytoplasm, suggesting that the threshold free calcium required to trigger calcium-stimulated calcium release might be pH dependent. The results of injection of pH-buffered calcium buffers support this conjecture: for a tenfold increase in hydrogen ion concentration, free calcium must also be raised tenfold to elicit the calcium transient.     

Calcium ionophore-induced egg activation and apoptosis are associated with the generation of intracellular hydrogen peroxide

The present study was designed to investigate whether calcium ionophore-induced activation and apoptosis are associated with the generation of hydrogen peroxide (H(2)O(2)) in rat eggs cultured in vitro. Culture of metaphase-II (M-II) arrested eggs in Ca(2+)/Mg(2+)-deficient medium did not induce egg activation, while a second polar body was observed in 20% of eggs when cultured in Ca(2+)/Mg(2+)-supplemented medium. In Ca(2+)/Mg(2+)-deficient medium, lower concentrations of calcium ionophore (0.2,0.4 and 0.8 microm) not only induced egg activation in a dose-dependent manner but also generation of intracellular H(2)O(2) (84.40+/-0.50 ng/egg) when compared to control eggs (80.46+/-1.34 ng/egg). The higher concentration of calcium ionophore (1.6 microm) induced apoptosis and pronounced generation of intracellular H(2)O(2) (92.43+/-0.93 ng/egg) in treated eggs. Conversely, cell-permeant antioxidant such as 2(3)-tert-butyl-4-hydroxyanisole (BHA) reduced intracellular H(2)O(2) level (81.20+/-1.42 ng/egg) and protected against calcium ionophore-induced morphological changes characteristics of egg activation and apoptosis. These results clearly suggest that calcium ionophore-induced activation and apoptosis are associated with the generation of intracellular H(2)O(2) in rat eggs.     

eIF4E‐Binding proteins are differentially modified after ammonia versus intracellular calcium activation of sea urchin unfertilized eggs

Fertilization of sea urchin eggs triggers a rise of protein synthesis mainly dependent on the cap‐binding protein eIF4E, which is released from its repressor 4E‐BP and associates with eIF4G. Association of eIF4G with eIF4E is a crucial event for the onset of the first mitotic division following fertilization. Artificial activation of unfertilized eggs with the calcium ionophore A23187 results in the activation of protein synthesis comparable to the one triggered by fertilization, while increasing the intracellular pH by ammonia treatment results in partial activation of protein synthesis. Nevertheless, artificial activation does not induce the mitotic division. Here we investigate the effect of calcium ionophore and ammonia treatment of unfertilized eggs on eIF4E and its two antagonist partners, 4E‐BP and eIF4G. We show that the addition of calcium ionophore to unfertilized eggs induces permanent dissociation between eIF4E and 4E‐BP, whereas a reversible dissociation of the complex occurs after ammonia treatment. The regulation of the complex correlates with permanent or reversible 4E‐BP disappearance depending on the treatment used to trigger artificial activation. Furthermore, while calcium ionophore treatment of unfertilized eggs induces eIF4G modifications comparable to those observed following fertilization, ammonia treatment does not. These results suggest that ionophore and ammonia treatments of unfertilized eggs induce differential protein synthesis activation by targeting eIF4E availability and specific regulation through its two partners 4E‐BP and eIF4G. Mol. Reprod. Dev. 77: 83–91, 2010. © 2009 Wiley‐Liss, Inc.     

Calcium ultrastructural localization in Xenopus laevis eggs following activation by pricking or by calcium ionophore A 23187

In the egg of Xenopus laevis a cortical network of smooth endoplasmic reticulum (SER) surrounds and interconnects each cortical granule (CG) (Campanella and Andreuccetti, '77). This network is a possible intracellular site of calcium storage to be called into action for CG exocytosis. In our experiments, Xenopus eggs, unfertilized or activated by pricking or by calcium ionophore A 23187, have been fixed in osmium-pyroantimonate for calcium localization. Our data show that deposits can be detected only in activated eggs. The calcium chelator edetate (EGTA) and x-ray microprobe analysis demonstrate that they contain calcium. Deposits are found on liposomes and on all intraovular cytomembranes, which therefore appear to be possible sites of calcium sequestration. In the case of ionophore-activated eggs, deposits are detectable independently of the presence of extracellular calcium. These data show that in Xenopus at activation an intracellular liberation of calcium occurs similar to that described in other species. Furthermore, the fact that antimony deposits are observed only after activation makes Xenopus eggs appropriate material in which to follow the temporal and spatial sequence of appearance of the deposits during the early stages of activation. Our results show that antimony deposits appear first in SER vesicles between the plasma membrane and CGs and then spread to the rest of the egg cytomembranes. These data corroborate our hypothesis that in Xenopus the cortical SER network is the first intracellular site where calcium is released at activation. The possible mechanism of calcium release and propagation along the egg cortex is discussed.     

Signal transduction at fertilization: the Ca2+ release pathway in echinoderms and other invertebrate deuterostomes

Gamete interaction and fusion triggers a number of events that lead to egg activation and development of a new organism. A key event at fertilization is the rise in intracellular calcium. In deuterostomes, this calcium is released from the egg's endoplasmic reticulum and is necessary for proper activation. This article reviews recent data regarding how gamete interaction triggers the initial calcium release, focusing on the echinoderms (invertebrate deuterostomes) as model systems. In eggs of these animals, Src-type kinases and phospholipase C-gamma are required components of the initial calcium trigger pathway in eggs.     

Distinctive subcellular alterations induced by hypertonic stress in sea urchin eggs

Sea urchin eggs continuously exposed to a hypertonic solution were ultrastructurally examined for osmotic-stress induced alterations. No fertilization membranes formed during the treatment and the surface-cortex complexes remained unaltered from the unfertilized state. However, the osmotic stress did induce a number of subcellular changes. During the first 30 minutes of the treatment the eggs formed many endoplasmic reticulum whorls and compacted Golgi body aggregations. Both of these new formations can be correlated with rapid changes in intracellular calcium, known to occur in hypertonic stressed eggs. Aggregations of mitochondria could be observed at later stages; these aggregations can also be related to subcellular stress and possible changes in internal calcium concentrations. The various morphological transitions within the cytoplasm, along with the lack of a cortical reaction in these eggs, not only supports the idea that calcium is released during parthenogenetic activation, but also suggests that this free calcium originates from stores other than the stores that are involved during fertilization or simple artificial activation.     

The dynamics of calcium oscillations that activate mammalian eggs

It has been known for some time that mammalian eggs are activated by a series of intracellular calcium oscillations that occur shortly after sperm egg membrane fusion. Recent work has identified a novel sperm specific phospholipase C zeta as the likely agent that stimulates the calcium oscillations in eggs after sperm-egg membrane fusion. PLCzeta is stimulated by low intracellular calcium levels in a manner which suggests that there is a regenerative feedback of calcium release and PLCzeta induced inositol 1,4,5-trisphophate (InsP(3)) production in eggs. This implies calcium oscillations in fertilizing mammalian eggs are driven by underlying oscillations of InsP(3). This model of oscillations is supported by the response of mouse eggs to sudden increases in InsP(3). The cellular targets of calcium oscillations include calmodulin-dependent protein kinases, protein kinase C and mitochondria. There is evidence that eggs might be best activated by multiple calcium increases rather than a single calcium rise. As yet we do not fully understand how the target of calcium in a mammalian egg might decode the patterns of calcium changes that can occur during egg activation.     

Intracellular calcium release at fertilization in the sea urchin egg.

Fertilization or ionophore activation of Lytechinus pictus eggs can be monitored after injection with the Ca-sensitive photoprotein aequorin to estimate calcium release during activation. We estimate the peak calcium transient to reach concentrations of 2.5–4.5 μM free calcium 45–60 sec after activation and to last 2$\text{3}\text{?}$ min, assuming equal Ca²⁺ release throughout the cytoplasm. Calcium is released from an intracellular store, since similar responses are obtained during fertilization at a wide range of external calcium concentrations or in zerocalcium seawater in ionophore activations. In another effort to estimate free calcium at fertilization, we isolated egg cortices, added back calcium quantitatively, and fixed for observation with a scanning electron microscope. In this way, we determined that the threshold for discharge of the cortical granules is between 9 and 18 μM Ca²⁺. Therefore, the threshold for the in vitro cortical reaction is about five times the amount of free calcium, assuming equal distribution in the egg. This result suggests that transient calcium release is confined to the inner subsurface of the egg.     

Activation of sea urchin eggs by a channel-forming ionophore amphotericin B

Micromolar amounts of the channel-forming ionophore amphotericin B (AMB) can activate Hemicentrotus pulcherimus eggs. Except for protein synthesis, activation by AMB was fairly normal in artificial sea water. In the case of AMB activation, it was found that external Na⁺ is necessary to initiate the activation process. External Ca²⁺, however, is not necessarily required—other than for complete fertilization membrane formation. These results suggest that AMB may induce the release of Ca²⁺ required for activation from internal calcium stock, as has been reported previously by many authors, using A 23187.     

Hydrogen peroxide induces Src family tyrosine kinase-dependent activation of Xenopus eggs

Fertilization is accompanied by a rapid and transient calcium release in eggs, which is required for the onset of zygotic developmental program or 'egg activation'. Recently, it was found that Src family tyrosine kinase (SFK)-dependent phospholipase C (PLC) activity is necessary for the calcium transience in fertilized Xenopus eggs. The present study demonstrates that hydrogen peroxide (H2O2) stimulates protein-tyrosine phosphorylation in Xenopus eggs, which occurs primarily in the egg cortex of the animal hemisphere as revealed by indirect immunofluorescence study. Egg SFK was found to be upregulated by H2O2 while the SFK-specific inhibitor PP1 effectively blocked H2O2-induced tyrosine phosphorylation. As in fertilized eggs, PLCgamma, but not Shc, was tyrosine-phosphorylated in H2O2-treated eggs. H2O2 also caused inositol 1,4,5-trisphosphate (IP3) production and sustained calcium release. After limited application of H2O2, elevated SFK activity and tyrosine phosphorylation were quickly reversed. Under such conditions, eggs showed cortical contraction and dephosphorylation of p42 MAP kinase, both of which are indicative of egg activation. These egg activation events, as well as H2O2-induced IP3 production and calcium release, were sensitive to PP1 and PLC inhibitor U-73122. Together, the present study demonstrated that H2O2 can mimic, at least in part, early events of Xenopus egg activation that require an SFK-dependent PLC pathway.     

Protein kinase C acts downstream of calcium at entry into the first mitotic interphase of Xenopus laevis.

Transit into interphase of the first mitotic cell cycle in amphibian eggs is a process referred to as activation and is accompanied by an increase in intracellular free calcium [( Ca2+]i), which may be transduced into cytoplasmic events characteristic of interphase by protein kinase C (PKC). To investigate the respective roles of [Ca2+]i and PKC in Xenopus laevis egg activation, the calcium signal was blocked by microinjection of the calcium chelator BAPTA, or the activity of PKC was blocked by PKC inhibitors sphingosine or H7. Eggs were then challenged for activation by treatment with either calcium ionophore A23187 or the PKC activator PMA. BAPTA prevented cortical contraction, cortical granule exocytosis, and cleavage furrow formation in eggs challenged with A23187 but not with PMA. In contrast, sphingosine and H7 inhibited cortical granule exocytosis, cortical contraction, and cleavage furrow formation in eggs challenged with either A23187 or PMA. Measurement of egg [Ca2+]i with calcium-sensitive electrodes demonstrated that PMA treatment does not increase egg [Ca2+]i in BAPTA-injected eggs. Further, PMA does not increase [Ca2+]i in eggs that have not been injected with BAPTA. These results show that PKC acts downstream of the [Ca2+]i increase to induce cytoplasmic events of the first Xenopus mitotic cell cycle.     

Mechanical stimulation by osmotic and hydrostatic pressure activates Drosophila oocytes in vitro in a calcium-dependent manner

Embryogenesis in vertebrates and marine invertebrates begins when a mature oocyte is fertilized, resulting in a rise in intracellular calcium (Ca²⁺) that activates development. Insect eggs activate without fertilization via an unknown signal imparted to the egg during ovulation or egg laying. One hypothesis for the activating signal is that deformation of eggs as they pass through a tight orifice provides a mechanical stimulus to trigger activation. Ovulation could produce two forms of mechanical stimulus: external pressure resulting from the passage of oocytes from the ovary into the narrow oviducts, and osmotic pressure caused by hydration-induced swelling of the oocyte within the oviducts. Ovulation could also trigger activation by placing the oocyte in a new environment that contains an activating substance, such as a particular ion. Here, we provide the first evidence that Drosophila oocytes require Ca²⁺ for activation, and that activation can be triggered in vitro by mechanical stimuli, specifically osmotic and hydrostatic pressure. Our results suggest that activation in Drosophila is triggered by a mechanosensitive process that allows external Ca²⁺ to enter the oocyte and drive the events of activation. This will allow exploitation of Drosophila genetics to dissect molecular pathways involving Ca²⁺ and the activation of development.     

Biochemical and biological effects of KN-93, an inhibitor of calmodulin-dependent protein kinase II, on the initial events of mouse egg activation induced by ethanol.

Calmodulin-dependent protein kinase II (CaMKII) is transiently activated in mouse eggs by the increase in calcium that occurs upon activation with ethanol. This study investigated the biological and biochemical effects of KN-93, a reported selective inhibitor of CaMKII, to explore the potential role of this kinase in the initial events of egg activation. Mouse eggs were incubated for 30 min in the presence of different concentrations of KN-93 and induced to activate by 7% ethanol. KN-93 elicited a dose-dependent inhibition of polar body emission that resulted from the failure of the eggs to undergo meiosis resumption and inactivation of maturation-promoting factor (MPF). Furthermore, 15 mumol KN-93 l-1 produced a marked reduction in ethanol-induced loss of cortical granules. In vivo biochemical analysis revealed that 15 mumol KN-93 l-1 was responsible for significant inhibition of ethanol-stimulated CaMKII. The activity of the enzyme remained at a resting value, in spite of the presence of a calcium signal similar to that measured in control activated eggs. The inhibitory effects of KN-93 on the parameters tested in this study could not be mimicked by the inactive analogue KN-92. These results show that in mouse eggs, when ethanol-induced CaMKII activation was prevented, cortical granule exocytosis and meiosis resumption were inhibited. This suggests that CaMKII acts as a switch in the transduction of the calcium signal triggering mammalian egg activation.