Ca2+-induced change of dorsal-ventral polarity in frog (Rana temporaria) eggs

Abstract. The effect of local Ca²⁺ administration 10–20 min after fertilization and during artificial activation was examined in Rana temporaria eggs. Ca²⁺ was injected into the pigmented region near the boundary between pigmented and unpigmented domains. The locations of egg gray crescent (GC) and dorsal lip of blastopore (DLB), as predictors of the dorsal region in embryos, as well as the measurements of angles between GC middle and sperm entry site were observed. In more than 70% of the cases, microinjection of Ca^{2 +} into subcortical cytoplasm and egg pricking in high-Ca^{2 +} solutions induced GC and DLB formation near the injection site. The formation of Ca²⁺-induced GC occurred mostly as in control eggs. In addition premature displacement of the egg surface was observed near the prick site in high-Ca^{2 +} solutions. GC formation occurred by displacement of the pigmented surface in the same direction as earlier wound translocations. These results show that Ca²⁺ injection determines the direction of the surface movement.     

Fertilization envelope formation induced by melittin in sea urchin eggs does not depend on Ca2+ signalling

In sea urchin eggs, 10 μg/mL melittin was found to induce fertilization envelope formation without any increase in [Ca²⁺]_i (the intracellular free Ca²⁺ level). On the other hand, 10 μmol/L Br-A23187 and 100 μg/mL SDS induced fertilization envelope formation associated with [Ca²⁺]_i increase. If EGTA was injected into eggs to make an intracellular concentration of 2 mmol/L, [Ca²⁺]_i became quite low and was not altered by melittin, or by Br-A23187 and SDS. In eggs containing EGTA, fertilization envelope formation was induced by melittin even in Ca²⁺-free artificial sea water, but not by Br-A23187 or SDS. Thus [Ca²⁺]_i is essential for induction of a fertilization envelope in sea urchin eggs by Br-A23187 or SDS but not by melittin. Melittin probably activates some Ca²⁺-independent reaction downstream of Ca²⁺-dependent reactions in a sequential reaction system that finally results in fertilization envelope formation.     

The Wave Pattern of Free Calcium Release Upon Fertilization in Medaka and Sand Dollar Eggs

A transient rise in the concentration of Ca²⁺ in the cortex upon fertilization was demonstrated in medaka eggs injected with aequorin. Detection of the aequorin luminescence with an ultra-high sensitivity photonic microscope system revealed a wave of increased Ca²⁺ concentration starting at the site of sperm entry (animal pole) and being propagated along the cortex of the egg toward the antipode. The wave traversed the entire egg surface within 2–3 min. The peak value of the aequorin luminescence, and therefore the peak value of the Ca²⁺ transient, was generally higher at the site of sperm entry than in other regions. The peak values of the luminescence (and therefore of the Ca²⁺ concentration in the cortex) remained fairly constant during propagation of the wave. Microinjection of Ca²⁺ into the cortex also induced a Ca²⁺ wave. When the egg was stimulated by microinjection of Ca²⁺ at the equatorial region, the Ca²⁺ wave was propagated at a fairly constant speed over the egg surface, except at the region near the vegetal pole where the wave was retarded. Simultaneous recording of the Ca²⁺ wave and the wave of cortical change (breakdown of cortical alveoli) in eggs during fertilization revealed that the Ca²⁺ wave preceded the wave of cortical change.
A Ca²⁺ wave was also demonstrated in sand dollar eggs, although due to their smaller size the phenomenon was not as clear as in medaka eggs.     

Ca2+ release from intracellular stores by thapsigargin in sea urchin eggs: Relationship to larval development and relevance in egg activation

Thapsigargin (Tg), an inhibitor of microsomal Ca²⁺ ATPase, is used as a tool to study the changes in Ca²⁺ sequestration in sea urchin eggs and their relationship to embryonic development. Micromolar amounts of Tg inhibit ATP-dependent Ca²⁺ sequestration in a dose-dependent and non-reversible manner, depending on the bulk of biological material used. IC_5O values are 1 nmol/L and 1–10μmol/L, respectively, in the cortical Ca²⁺ stores (isolated cortices preparation) and in digitonin-permeabilized eggs, a preparation giving access to the deeper reticulum compartment. Micromolar Tg does not induce Ca²⁺ release from ⁴⁵Ca pre-loaded cortices but leads to a loss of 25% of the total Ca²⁺ content from the cortical area. Using microspectrofluorimetry of fura-2-loaded eggs, we found that 10 μmol/L Tg induced a moderate rise in cytosolic Ca²⁺ activity as compared with the fertilization-induced Ca²⁺ transient whether eggs were fertilized or not. Early events related to fertilization as, for example, elevation of the fertilization envelope, proton excretion and sustained increase of amino acid uptake, are triggered by 10μmol/L Tg but with a delayed onset relative to sperm-induced effects. The present findings indicate that although it triggers most fertilization-related events, Tg cannot be considered as a true mitotic agent in sea urchin eggs. When added after fertilization, Tg affects cleavage and the further embryonic development giving rise to abnormalities comparable to the animalized larvae obtained with other compounds responsible for the inhibition of reticular Ca²⁺ sequestration.     

Sea urchin fertilization stimulates CaM kinase-II (multifunctional [type II] Ca2+/CaM kinase) activity and association with p34cdc2

Upon fertilization, the sea urchin egg synthesizes proteins which impart a Ca²⁺ dependence to M-phase onset. A potential target of this Ca²⁺ dependence may be CaM kinase-II (the multifunctional [type II] Ca²⁺/calmodulin [CaM]-dependent protein kinase) which is necessary for nuclear envelope breakdown in fertilized sea urchin eggs. This study was intended to determine whether sea urchin CaMK-II is activated after fertilization and whether it interacts with other known M-phase regulators, such as p34^cdc2. We report that total CaMK-II activity, measured by solution assays, increases after fertilization, peaking just prior to cleavage. Interestingly, total CaMK-II activity continues to fluctuate, peaking again prior to second and third cleavage. Gel assays also reveal enhanced levels of the 56 and 62 kDa potential CaMK-II phosphoproteins after fertilization. Finally, CaMK-II activity and only the 62 kDa phosphoprotein physically associate with p34^cdc2, but again only after fertilization. These changes in CaMK-II activity and p34^cdc2-association after fertilization may ensure that Ca²⁺ signals are targeted to the M-phase machinery at the appropriate developmental times.     

Probable participation of phospholipase A2 reaction in the process of fertilization-induced activation of sea urchin eggs

In sea urchin eggs activated by sperm, A23187 or melittin, BPB (4-bromophenacyl bromide, a phospholipase A₂ inhibitor) blocked fertilization envelope formation and transient CN⁻-insensitive respiration in a concentration-dependent manner. BPB had virtually no effect on the increase in [Ca²⁺]_i, (cytosolic Ca²⁺ level), the activity of phosphorylase a and the rate of protein synthesis, as well as acid production and augmentation of CN⁻-sensitive respiration. BPB also inhibited fertilization envelope formation and augmentation of CN⁻-insensitive respiration induced by melittin. Melittin, known to be an activator of phospholipase A₂, induced the envelope formation, acid production, augmentation of CN⁻-insensitive and sensitive respiration, but did not cause any increase in [Ca²⁺]_i, the phosphorylase a activity and the rate of protein synthesis. An activation of phospholipase A₂ induced by Ca²⁺ or melittin seems to result in cortical vesicle discharge and production of fatty acids, which are to be utilized in CN⁻-insensitive lipid peroxidase reactions. Activation of other examined cell functions in eggs activated by sperm or A23187, probably results from Ca²⁺-triggered sequential reactions other than Ca²⁺-caused activation of phospholipase A₂.     

Change in the Respiratory Rate of Sea Urchin Spermatozoa following Sperm-egg Interaction in the Absence of Mg2+

Spermatozoa of the sea urchin, Hemicentrotus pulcherrimus (10⁸ cells/ml), preincubated with unfertilized eggs deprived of jelly coats (more than l0⁵ cells/ml) at 20°C for 20min in Mg²⁺ free artificial sea water containing 1 mM Ca²⁺ (MFASW), exhibited very low respiration, which was enhanced by 2, 4 dinitrophenol (DNP). The fertilization rate in MFASW was usually less than 5% and was about 25% at most. Preincubation with fertilized eggs (with and without a fertilization membrane) in MFASW did not reduced the respiratory rate of spermatozoa. The rate of sperm respiration was lower in MFASW than in artificial sea water (ASW), but was higher than the respiratory rate of spermatozoa preincubated in MFASW with unfertilized eggs. Sperm respiration in MFASW or in ASW was not stimulated by 2, 4 dinitrophenol. Almost complete inhibition of sperm respiration was obtained with unfertilized eggs fixed with glutaraldehyde at concentrations of above 10⁵ cells/ml in MFASW and of about l0⁴ cells/ml in ASW. The respiratory rate of spermatozoa treated with fixed eggs was enhanced by DNP. It is concluded that the respiratory rate of the spermatozoa is reduced by their interaction with unfertilized eggs before their penetration into the eggs.     

Vegetalization Induced by Procaine and Tetracaine in Sea Urchin Embryos

Vegetalization of sea urchin embryos was induced by the treatment with procaine and tetracaine, inhibitors of Ca²⁺mobilization, for 3 hr starting 3–5 hr after insemination at 20°C. The treatment starting 7 hr after insemination sometimes produced similar type of vegetalized embryos. The pulse treatment starting at the other stages hardly yielded vegetalized embryos. The stages at which these compounds were effective to produce vegetalized embryos were almost the same to those for Li⁺to make embryos vegetalized. On the basis of known inhibitory effects of tetracaine, procaine and Li⁺on Ca²⁺mobilization, we postulate that Ca²⁺dependent reactions participate in the process of cell determination at these stages. Inhibitory effects of procaine, tetracaine and Li⁺on Ca²⁺dependent induction of fertilization membrane formation, found in the present study, indicate that these compounds block Ca²⁺mobilization in sea urchin eggs.     

Requirement of Extracellular Calcium Ions for the Early Fertilization Events in the Medaka Egg

The requirement for calcium and the change in calcium content in eggs of Oryzias iatipes during the cortical reaction and sperm penetration were examined. Naked eggs failed to exhibit the cortical reaction upon insemination under Ca Mg-free conditions. These eggs exhibited the cortical reaction by reinsemination in the presence of extracellular Ca²⁺. The effect of extracellular Ca²⁺ on sperm penetration could be replaced by one of several divalent cations in the external medium. Unlike the cortical reaction, sperm penetration failed to be induced by microinjection to increase intracellular Ca²⁺. Verapamil significantly reduced the action of extracellular Ca²⁺ or Ba²⁺ of divalent cations examined in fertilization, while TEA and TTX had no effect on fertilization in the presence of these cations. No ⁴⁵Ca uptake into the egg proper was recognized before completion of the cortical reaction. These observations suggest that extracellular divalent cations are indispensable for sperm stimulation of the egg and its penetration into the egg, for which an influx of Ca²⁺ from the external medium is not required.     

Fertilization Reaction without Changes in Intracellular Ca2+ in Medaka Eggs—An Experiment with Acetone-Treated Eggs

To investigate whether or not causal relationship exists between the increase in intracellular Ca²⁺ and other cortical reactions at fertilization in the medaka, Oryzias latipes , intracellular Ca²⁺ was determined from luminescence of aequorin previously microinjected into cortical cytoplasm in acetone-treated eggs, when they were inseminated or activated by microinjection of Ca²⁺. Neither an increase in cytoplasmic calcium nor exocytosis of cortical alveoli occurred in eggs treated with acetone, though other events of fertilization i.e. completion of meiosis, fusion of pronuclei, and accumulation of cortical cytoplasm with intact cortical alveoli in the animal pole region were observed in normal time sequence in these eggs. When denuded eggs were treated with acetone, contraction of the egg and slow resumption of meiosis (extrusion of polar body) were observed without insemination. When denuded eggs were inseminated immediately after acetone-treatment, the number of spermatozoa that penetrated into the egg was greater in the animal hemisphere than in the vegetal hemisphere. These results may indicate that acetone inactivates the egg plasma membrane or its adjacent cortical cytoplasm so that it cannot participate in a propagative increase in intracellular Ca²⁺ and exocytosis, while it also induces cytoplasmic activation leading to egg contraction, resumption of meiosis and formation of pronuclei. The present results suggest that sperm penetration, resumption of meiosis and ooplasmic segregation are regulated separately from the release of intracellular Ca²⁺ and exocytosis.     

Cytochemical Ca2+ Distribution in Activated Discoglossus pictus Eggs: A Gradient in the Predetermined Site of Fertilization

The K-pyroantimonate/OsO₄ (PA) cytochemical method coupled with EGTA and X-ray microanalytical controls has been used to localize Ca²⁺ at egg activation in Discoglossus pictus eggs. The results show that: 1) the PA method is able to selectively localize Ca²⁺ pools mobilized by activating stimuli; 2) the smooth endoplasmic reticulum (SER) elements located in the animal dimple region, i.e. in the predetermined site of fertilization, are the first egg components labeled by precipitates; 3) a decreasing gradient of precipitates is present from the center beyond the boundaries of the dimple region; 4) precipitates are lacking in the remainder of the egg even at late times after activation.
The possibilities are discussed that a) SER is the major Ca²⁺-releasing store at activation in Discoglossus , and b) the observed gradient of pyroantimonate-detected Ca²⁺ reflects an ionic Ca²⁺ gradient.     

Formation of the enveloping layer of the chum salmon egg in isotonic salt solutions

After stimulation in a hypotonic solution (9.4 mOsm kg⁻¹), inseminated eggs of the chum salmon Oncorhynchus keta initiate cleavages in isotonic salmon Ringer's solution (267.3 mOsm kg⁻¹) containing 3.2 mM Ca²⁺ ions. Blastomeres of these eggs, however, separate from each other and the enveloping layer is not observed at the blastula stage. An increase in external divalent cations rescues the separation; the concentration of CaCl₂ in the external medium should be 25 mM or more to induce close contact of blastomeres and the formation of an enveloping layer in isotonic salt solutions. The effectiveness of Ca²⁺ ions can be substituted by Mg²⁺, Sr²⁺ and Zn²⁺ ions; the same results are obtained in isotonic MgCl₂ and SrCl₂ solutions (100 mM) or in isotonic salmon Ringer's solution containing Zn ions (6.2 mM). The close contact of blastomeres and the formation of an enveloping layer are also observed in a low Ca²⁺ concentration (< 0.1 mM) in a hypotonic salt solution (9.4 mOsm kg⁻¹). The Ca²⁺ level in the external medium to induce the enveloping layer formation seems to be correlated with the salinity of the incubation medium. It is suggested that adhesion molecules on the surface of blastomeres in the chum salmon eggs are different in properties from those found in sea urchin and other fish species.     

Activation of Sea Urchin Eggs by Halothane and Its Inhibition by Dantrolene

Eggs of the sea urchin, Hemicentrotus pulcherrimus , were stimulated by halothane, known to induce Ca²⁺ release from sarcosome, to cause fertilization membrane formation in normal and Ca²⁺ free artificial sea water. In the absence of external Ca²⁺, halothane-induced formation of fertilization membrane was inhibited by dantrolene, an inhibitor of Ca²⁺ release from sarcosome, but was not blocked by nifedipine, a Ca²⁺ antagonist specific to Ca²⁺ channels in plasma membrane. Ca²⁺ release from sedimentable fraction isolated from eggs was induced by halothane and was inhibited by dantrolene, but was not blocked by nifedipine. In normal artificial sea water, halothane-caused egg activation was not inhibited either by dantrolene or by nifedipine, but was blocked in the presence of both compounds. ⁴⁵Ca²⁺ influx was substantially stimulated by halothane in eggs exposed to ⁴⁵CaCl₂. Halothane-induced ⁴⁵Ca²⁺ influx into eggs was inhibited by nifedipine but was not blocked by dantrolene. When Ca²⁺ release from intracellular organellae is blocked, Ca²⁺ transport through Ca²⁺ channels in plasma membrane probably acts as a "fail-safe" system to induce an increase in cytosolic Ca²⁺ level, resulting in egg activation.     

Role of cell adhesion in the specification of pigment cell lineage in embryos of the sea urchin, Hemicentrotus pulcherrimus

To clarify the role of cell adhesion in the specification of pigment cell lineage in sea urchin embryos, cell contacts were inhibited by Ca²⁺-free artificial seawater (ASW) treatment, and the number of differentiated pigment cells was examined by the method devised for the present study. Obtained results showed that inhibition of cell contacts during mid-to-late blastula stage greatly affects the number of pigment cells. Treatment with Ca²⁺-free ASW during 7.5–10.5h of development drastically decreased the number of pigment cells, indicating that cell adhesion during this period is indispensable for the specification of pigment cell lineage. On the other hand, the number of pigment cells were increased by the treatment during 9.5–12.5 h of development. It was suggested that this increase was caused by excess divisions of the precursor cells, that is, the division schedule of the precursor cells was altered by inhibition of cell contacts at this period. Interestingly, the number of pigment cells was a multiple of four in a majority of embryos in which pigment cells were drastically decreased in number. These findings suggest that the founder blastomeres of the pigment cell lineage are specified during 7–10 h of development, and that these blastomeres divide twice before they differentiate into pigment cells.     

Electron Microscopic Study of Development in a Sea Cucumber,Stichopus tremulus (Holothuroidea), from Unfertilized Egg through Hatched Blastula

In this, the first fine structural study of sea cucumber embryology, eggs and embryos of Stichopus tremulus developing at 7.5°C are described from spawning through hatched blastulae. Spawned eggs are at about first meiotic metaphase and are surrounded by a jelly layer that remains around the embryos until hatching. No vitelline coat can be demonstrated, but whether it is truly absent or removed by electron microscopic processing is not known. Insemination initiates a rapid cortical reaction, completed within 2 min., which involves a wave of cortical granule exocytosis and fertilization envelope formation. The compactly fibrous fertilization envelope is about 50 nm thick and appears to consist entirely of ejected cortical granule material (if one assumes that there is no vitelline coat). As the fertilization envelope elevates, no hyaline layer appears in the perivitelline space. The first and second polar bodies are emitted, respectively, at about 9 and 15 min. after insemination. The first seven or so cleavages are equal, radial, and occur approximately every 4 hr. The blastocoel opens up at the four-cell stage and, during the earlier cleavages, remains connected with the perivitelline space via numerous gaps between the roughly spherical blastomeres. At the 64-cell stage, these gaps begin to close as the blastomeres start to become cuboidal; in addition, an embryonic cuticle is produced on the apical surface of each blastomere. In embryos of several hundred cells, the blastomeres become associated apicolaterally by junctional complexes, each consisting of a zonula adherens and a septate junction. Several hours before hatching, a single cilium is produced at the apical surface of most blastomeres. At hatching (about 50 hr after insemination), the ciliated blastula leaves behind the fertilization envelope and jelly layer. Swimming blastulae soon begin to elongate in the animal-vegetal axis, and a basal lamina develops on blastomere surfaces facing the blastocoel. The discussion includes a fine structural comparison of egg coats among the five classes of the phylum Echinodermata.     

Activation of Xenopus Eggs by an Extract of Cynops Sperm

An extract obtained from Cynops sperm induced the activation of both Cynops and Xenopus eggs with accompanying changes in the potential of the egg membrane that were quite similar to those caused by the Cynops sperm. The activation-inducing properties of the extract were abolished by treatment with proteinase K or by heating (60°C, 15 min) and were associated with a protease activity against peptidyl Arg-MCA substrates. The activation of Xenopus eggs by the extract was inhibited by those substrates, or by protease inhibitors, aprotinin or leupeptin. The protease activity was localized in the acrosomal region of Cynops sperm. The activation of Xenopus eggs by the extract was prevented when the exterior concentration of Ca²⁺ions, [Ca²⁺]₀, was reduced to 1.5 μM, but it was enhanced when [Ca²⁺]₀ was increased to 340 μM. The activation of Xenopus eggs by the extract was not affected by positive clamping when [Ca²⁺]₀ was 340 μM. These results suggest that the sperm extract contains a protease that causes an increase in the influx of Ca²⁺ions that results in voltage-insensitive activation of the egg.     

EFFECTS OF TAURODEOXYCHOLATE AND CALCIUM ON CONTRACTILITY OF NEWT EGGS

Injection of taurodeoxycholate (TDOC) alone or in combination with 10⁻⁵ M Ca²⁺(Ca-EGTA buffer) into newt eggs induced a cup- or furrow-like depression on the egg surface. Reduction of the Ca²⁺ concentration inhibited the response. These findings imply that TDOC induces a cytoplasmic contraction associated with the membrane and that a micromolar Ca²⁺ concentration is required for this process. Injection of 10⁻⁵ M Ca²⁺(Ca-EGTA buffer) alone had no effect. On the basis of these findings, the roles of TDOC and Ca²⁺ in induction of contraction were discussed.     

Fertilization Membrane Formation in Sea Urchin Eggs Induced by Drugs Known to Cause Ca2+Release from Isolated Sarcoplasmic Reticulum

Ryanodine, miconazole, clotrimazole, doxorubicin, quercetin, halothane, caffeine and chloroform, which activate Ca²⁺-induced Ca²⁺release from Ca²⁺stores, induced Ca²⁺release from a particulate fraction isolated from sea urchin eggs, Ca²⁺influx into eggs and formation of a fertilization membrane in an appreciable number of eggs. Their minimum effective concentrations for inducing a fertilization membrane increased in the order of these drugs listed above, and this order was also the same as that of their minimum effective concentrations for inducing Ca²⁺release from the isolated particulate fraction. Their effect in inducing a fertilization membrane was blocked by ruthenium red and procaine, which inhibit Ca²⁺release from Ca²⁺stores. Thus these drugs probably induced sufficient Ca²⁺release to make the cytosolic Ca²⁺level high enough in many eggs for formation of a fertilization membrane. In the absence of external Ca²⁺, fewer eggs treated with these drugs formed a fertilization membrane and more eggs did so on further treatment with either A23187 or carbonylcyanide-p-trifluoromethoxy-phenylhydrazone (FCCP). Thus, a high level of Ca²⁺is probably derived from Ca²⁺release through Ca²⁺releasing channels (by A23187), from mitochondria (by FCCP) and its transport from the external medium.     

Changes in ion content and transport during development of embryonic rainbow trout

Wet mass and water content of four lots of whole eggs did not change throughout embryonic development of rainbow trout Oncorhynchus mykiss. Eggs in all four lots accumulated Na⁺. Eggs in lots 2 and 4 also accumulated Ca²⁺ and Cl^-, whereas eggs in lot 1 showed no significant change in Ca²⁺ or Cl^- and eggs in lot 3 showed no change in Cl^-and a small loss of Ca²⁺. Although the Na⁺ content of embryonic tissues increases in the later stages of development, the yolk sac content remained constant, indicating uptake of Na⁺ from the environment. Na+ uptake by whole eggs was non-saturable, consistent with diffusion of Na⁺ across the chorion into the perivitelline fluid. Na⁺ uptake in dechorionated embryos was saturable, as was Ca²⁺ uptake by both whole eggs and dechorionated embryos, consistent with active uptake or facilitated diffusion mechanisms at the surface of embryos. Very low Ca²⁺ uptake rates in dechorionated embryos suggest that the Ca²⁺ uptake mechanism is not fully developed until after hatching.     

Activation of newt eggs in the absence of Ca2+ activity by treatment with cycloheximide or D2O

Unfertilized eggs of urodeles that exhibit physiological polyspermy are difficult to activate by ordinary egg-activating agents, such as pricking and Ca²⁺ ionophores, that easily activate monospermic anuran eggs. Therefore, we have tested the effects of other agents that cause egg activation in non-amphibian species in order to investigate the mechanism of egg activation in urodeles. We have found that cycloheximide (a protein synthesis inhibitor), D₂O (that induces microtubule polymerization) and 6-DMAP (a protein kinase inhibitor) caused activation of unfertilized eggs of the newt, Cynops pyrrhogaster . The cell cycle, arrested at meiotic metaphase II, was resumed to form the second polar body accompanied by a loss of maturation promoting factor and cytostatic factor activity. The treated eggs underwent abnormal cleavage. These results indicate that protein synthesis followed by protein phosphorylation is necessary to maintain M phase in unfertilized Cynops eggs. Unfertilized eggs failed to be activated by pricking, but were activated by the ionophore A23187, but only at a concentration 30 times higher than that required to activate Xenopus eggs. Eggs whose intracellular Ca²⁺ ions had been chelated by BAPTA could also be activated by either cycloheximide or D₂O. Cycloheximide- as well as 6-DMAP-induced egg activations were not inhibited by nocodazole, a microtubule-depolymerizing agent. These results suggest that the inhibition of synthesis and phosphorylation of short-lived proteins acts as an egg activation mechanism, downstream of the site of Ca²⁺ action and independently of microtubule polymerization.