Experimental activation of ascidian eggs.

The effects of the ionophore A23187 on the activation of the eggs of Ascidia malaca have been studied. No common external ion in the sea water is found to be essential for the activation but lanthanum and manganese inhibit the response. These observations support the interpretation that activation of these eggs results from changes in free intracellular calcium levels. This has led to the prediction of two other activating treatments, namely high external calcium and addition of theophylline.     

In vitro activation of Drosophila eggs

Mature ovarian eggs of Drosophila can be activated by treatment with hypotonic buffers. Well-fed, 4-day-old virgin flies contain large numbers of partially dehydrated mature eggs. When these eggs are transferred to a hypotonic culture medium, the ovarian eggs swell immediately and within minutes up to 70% become impermeable. The following cellular events ensue: meiosis, which had been arrested at metaphase I, is completed and the "polar body" nuclei fuse; cortical multivesicular bodies with acid phosphatase activity appear within minutes; polar granules fragment, dissociate from mitochondria, and become associated with polysomes; finally, monosomal ribosomes move to the polysomal region of a sucrose gradient. Each of these events corresponds to the normal in vivo effects of ovulation of oocytes, whether or not they are fertilized. When ovarian eggs of a parthenogenetic strain of D. mercatorum were activated by hypotonic treatment, some eggs developed into normal embryos. The presence of high potassium, low pH, and polyethylene glycol enhanced the frequency of normal development. Thus, we suggest that the rehydration of mature oocytes, as they move from the ovary to the uterus, activates the maternal program of the oocyte.     

Partial activation of sea-urchin eggs by bonellin

We here describe further studies on the action of bonellin on sea-urchin eggs. Bonellin brings about Some of the changes that are known to occur in the egg upon fertilization. In particular, it appears to cause the increased rate of incorporation of amino acids into proteins, the increase of the voltage noise, and the exocytosis of some of the cortical granules. A comparison with the effect of ammonia is discussed.     

Cortical activity in vertebrate eggs. I: The activation waves

We present a physical model for the propagation of chemical and mechanical waves on the surface of vertebrate eggs. As a first step we analyzed the propagation of the calcium wave observed to sweep over the surface of the Medaka egg (Gilkey et al., 1978). It has been assumed that this wave is driven by a mechanism of calcium-stimulated-calcium-release. By formulating this hypothesis mathematically we can use the observed wavefront data to obtain a map of cortical reactivity. This map indicates a gradient of reactivity along the egg: highest in the animal hemisphere and tapering off towards the vegetal hemisphere. The cortex of Xenopus eggs is also capable of propagating a calcium wave (Busa & Nuccitelli, 1985). At about the same time a wave of expansion followed by a wave of contraction sweeps across the egg surface (Takeichi et al., 1984). We have proposed a mechanism for this wave pair based on the physical chemistry of actomyosin gels. The calcium wave activates solation factors which sever some of the actin chains which leads to an osmotic swelling of the gel. Calcium also activates the contractile machinery of the actomyosin system which causes the gel to contract. The contraction lags the swelling because of the nature of the kinetics: solation and swelling is a more rapid process than contraction. By writing the equations for gel expansion and contraction we can mimic the mechanical and chemical wave propagation by a computer simulation. If the model is correct this provides a method for using the waves as a diagnostic of the mechanochemical properties of the egg cortex.     

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.     

Developmental failure after experimental activation of insect eggs

Summary

Eggs taken from the genital tracts of Venturia (Nemeritis) canescens (Hym., Ichneum.) and Psychoda cinerea (Dipt., Psychod.) can be activated by brief exposure to distilled water. However, embryogenesis fails soon unless some other conditions are met. These are mechanical deformation of the egg in Venturia, and mating in Psychoda. Development ceases at or soon after meiosis in Psychoda and in most Venturia eggs; the remaining Venturia eggs fail to form an orderly blastoderm although they contain hundreds of nuclei. In Venturia the anomalies are probably due to insufficient activation of some cytoplasmic component(s) while Psychoda eggs fail in the absence of some paternal contribution, possibly the sperm which in this case must enter the oocyte within the follicle.     

Acid release following activation of surf clam (Spisula solidissima) eggs.

Acid release was observed after activation of Spisula eggs with excess KCI. This acid release begins within 20 sec after the activation and continues for 9–15 min. The amount of acid released was 6.8 μmole per milliliter of packed eggs. In Ca-free or Na-free sea water, the acid release is completely inhibited; subsequent addition of the deficient ion leads to acid release and breakdown of germinal vesicles. These results suggest that Spisula eggs release protons after activation in a manner similar to that of sea urchin eggs, and that acid release with concomitant increase in cytoplasmic pH is probably a general event on activation of marine eggs.     

Free calcium wave upon activation in Xenopus eggs

Eggs of Xenopus laevis were preloaded with aequorin and the spatial and temporal pattern of free calcium release in the egg cortex on artificial activation was determined by the aequorin luminescence emitted from the thin cortical layer of naturally opaque eggs. The aequorin luminescence was detected with a photonic microscope system consisting of a light microscope and a two-dimensional photon-counting system with an image processor. A free calcium increase was initiated around the point of prick activation. The state of increased Ca2+ propagated in the cortical cytoplasm of the egg as a wave with a velocity of about 8 micron/sec at 22 degrees C. This wave reached the antipode by 5 to 6 min of prick activation. The spatial pattern of the Ca2+ wave was similar to that of changes in brightness of the egg surface on activation, termed the "activation wave" by K. Hara and P. Tydeman (1979, Wilhelm Roux's Arch. Dev. Biol. 186, 91-94). To examine the temporal correlation between the Ca2+ wave and the activation wave, images of aequorin luminescence and those of the egg cortex taken by incident light illumination were recorded alternately in the same egg. The zone of free calcium increase corresponded to the light (relaxation) zone of the activation wave, where exocytosis of cortical granules and elongation of microvilli were taking place.     

Parthenogenetic activation decreases the polyphosphoinositide content of frog eggs

Polyphosphoinositides were quantified in metaphase II-arrested eggs of the amphibian Xenopus laevis and 8-10 min later in eggs activated by pricking. The content of phosphatidylinositol 4,5-biphosphate (PIP2) was remarkably high in metaphase II-arrested eggs with respect to that of phosphatidylinositol 4-phosphate (PIP). It was found to drop dramatically at activation. In contrast PIP content did not change significantly.     

Microvillar elongation following parthenogenetic activation of sea urchin eggs

Parthenogenetic activation of unfertilized sea urchin eggs with ammonium chloride at pH 8.0 resulted in a slow, but dramatic, reorganization of surface microvilli in four species of sea urchin eggs. Following NH4Cl treatment, elongation of microvilli on the egg surface was observed concomitant with the formation of microfilament bundles within the microvillar cores. A minimum of 2 h of treatment was required for elongation and microfilament bundle formation to occur. The maintenance of elongated microvilli was pH-sensitive; removal of the activating agent resulted in the retraction of extended microvilli while readdition of NH4Cl caused microvilli to elongate again. Accompanying microvillar elongation in activated eggs, there was an increased calcium uptake as measured by 45Ca uptake. Blocking calcium uptake by incubation in lanthanum chloride or zero-calcium seawater containing 2 mM EGTA prevented microvillar elongation. These results suggested that elongation of microvilli following parthenogenetic activation by NH4Cl is pH- and calcium-dependent and is similar to that observed during normal fertilization.     

Increased uptake of thymidine in the activation of sea urchin eggs. III. Effects of aphidicolin

Uptake and phosphorylation of externally supplied [3H]-thymidine are fully stimulated in fertilized sea urchin eggs exposed to 5.0 micrograms/ml aphidicolin. As in untreated controls, the rate of uptake in aphidicolin-treated eggs increases greater than 50-fold shortly after fertilization, and greater than 85% of the transported thymidine is immediately phosphorylated to the triphosphate. The intracellular levels of [3H]-thymidine triphosphate (3H-dTTP) resulting from an external supply of [3H]-thymidine is therefore equal in aphidicolin-treated and untreated fertilized eggs. Under the same experimental conditions, the incorporation of externally supplied [3H]-thymidine into newly synthesized DNA of fertilized eggs is 90% inhibited by exposure to aphidicolin. The full availability of 3H-dTTP in these eggs further suggests that aphidicolin inhibits specifically at the level of DNA synthesis. This inhibitory effect is proportional to the concentration of aphidicolin between 0 and 5.0 micrograms/ml. In the continuous presence of 5.0 micrograms/ml aphidicolin, fertilized eggs fail to undergo mitotic chromosome condensation, nuclear envelope breakdown, and cytokinesis, suggesting a dependent link between these processes and the completion of nuclear DNA synthesis.     

Methods for the activation of the resting eggs of Daphnia

SUMMARY. 1. The conditions required to initiate development of resting eggs of thirty-six clones of Daphnia representing seven species were investigated.
2. The temperature of both dark incubation and subsequent light treatment are shown to affect hatch success. By varying these parameters the majority of resting eggs from each test clone were stimulated to develop. Arctic clones required a low hatching temperature (7°C), whereas clones from warmer climates hatched best at 14–21°C.
3. Variation in hatching cues existed between conspecific individuals from different collection sites. These differences suggest that research determining macro- and microgeographic patterns in hatching phenology would be fruitful.     

Spontaneous activation of fish eggs is abolished by protease inhibitors

During spawning, eggs of most fish species entering the aquatic environment remain fertilizable for a relatively short period of time. This is due to the “spontaneous egg activation” giving rise to the fertilization membrane, which prevents the penetration of excessive and foreign sperm into the egg during normal fertilization. This work demonstrates that the fertilization membrane formation and the loss of fertilizability in aqueous solutions of different composition are inhibited by protease inhibitors, in particular, leupeptin and aprotinin. The presence of natural protease inhibitors in the ovarian fluid that prevent spontaneous egg activation is proposed. The decrease in the concentration of these inhibitors as the ovarian fluid is diluted in aquatic medium during spawning can explain egg activation in the absence of sperm.     

Primary and secondary messengers in the activation of ascidian eggs

Two early events of activation in the ascidian egg, the surface contraction and the fertilization current, were studied. Ca ionophore induces contraction without generating a fertilization current, whereas microinjection of IP3 or soluble fractions of homogenized spermatozoa trigger both a contraction and a current. This suggests that the primary trigger of activation in ascidian eggs is a soluble component of spermatozoa that may be released into the egg subsequent to gamete fusion. IP3, or other intermediates in phosphoinositide metabolism, is a putative second messenger that activates fertilization channels directly (probably a Ca-independent process), and subsequently induces surface contraction by releasing Ca from intracellular stores.     

Cortical modifications in Paracentrotus lividus eggs after ammonia activation

We have shown by electron microscopy that ammonia activation of Paracentrotus lividus eggs alters the inner ultrastructure of cortical granules. If activated eggs are inseminated, they fail to undergo a typical cortical reaction.     

Injection of a porcine sperm factor induces activation of mouse eggs

Injection of sperm preparations into mammalian oocytes and eggs has been shown to elicit persistent [Ca²⁺]i oscillations that closely resemble fertilization-associated Ca²⁺ release. However, the ability of these sperm fractions to initiate egg activation has not been clearly demonstrated. In the present experiments, mouse eggs injected with a porcine sperm preparation were evaluated for early and late events of activation. Events monitored included, among early events, the generation of [Ca²⁺]i oscillations and cortical granule exocytosis and, among late events, the decrease in histone H1 and myelin basic protein kinase activities, polar body extrusion, pronuclear formation, and cleavage to the two-cell stage. Injection of sperm fractions consistently evoked [Ca²⁺]i oscillations that, in turn, initiated all events of activation. Uninjected control eggs or eggs injected with buffer or heat-treated sperm fractions failed to show Ca²⁺ responses or activation. In addition, injection of sperm fractions into recently ovulated eggs (experiments were concluded within 15 hr after human chorionic gonadotropin administration) induced high rates of activation, while similarly aged eggs exposed to 7% ethanol for 5 min, a known parthenogenetic treatment, failed to activate. Together these results indicate that injection of sperm fractions elicits [Ca²⁺]i oscillations that are capable of initiating normal egg activation. These results support the hypothesis that a sperm component participates in the generation of fertilization-associated [Ca²⁺]i oscillations. Mol. Reprod. Dev. 49:37–47, 1998. © 1998 Wiley-Liss, Inc.     

Possible involvement of calmodulin in maturation and activation of Chaetopterus eggs

We report the isolation of calmodulin from oocytes of Chaetopterus pergamentaceus. The identification of this protein is based on (1) activation of beef heart cAMP phosphodiesterase, (2) heat stability, (3) sensitivity to chlorpromazine, and (4) electrophoretic mobility identical to that of porcine brain calmodulin after sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of either Ca2+ or EGTA. We treated oocytes with chlorpromazine and W-7 to investigate the involvement of calmodulin in meiosis initiation and egg activation. Very low concentrations of chlorpromazine inhibited germinal vesicle breakdown (GVBD). This effect was shown to be dependent upon bright indirect light, since the drug was much less effective at GVBD inhibition under conditions of very low illumination. Higher concentrations of chlorpromazine and W-7 (100 microM) inhibited GVBD and activated eggs with intact germinal vesicles as determined by fertilization envelope formation and the onset of ameboid activity. Neither egg activation nor inhibition of calmodulin stimulation of phosphodiesterase activity in vitro was affected by light. These results are consistent with a role for calmodulin in egg activation and GVBD, but suggest that chlorpromazine in bright light may prevent GVBD by some mechanism other than calmodulin inhibition.     

Catecholamine secretion and adenylate cyclase activation in sea urchin eggs

The role of neurotransmitters in sea-urchin eggs was investigated by studying their effect on adenylate cyclase of the egg membrane. Maximal stimulation of enzyme activity occurs in the presence of dopamine and GTP. 5-hydroxytriptamine, 5-methoxytriptamine and acetylcholine have no effect on activity, despite a decrease in intracellular cAMP level in eggs treated with 5-hydroxytriptamine antagonists as previously reported (Renaud et al., 1983). High-performance liquid chromatography (HPLC) revealed that dopamine is released from the sea-urchin egg into the external medium following fertilization.