Increased uptake of thymidine in the activation of sea urchin eggs. II. Cooperativity with phosphorylation, involvement of the cortex, and partial localization of the kinases

Uptake and phosphorylation of exogenously supplied thymidine are stimulated in Strongylocentrotus purpuratus eggs after fertilization. Before fertilization, the rate of uptake is low and less than 10% of the thymidine entering the egg is phosphorylated. After fertilization, the rate of uptake increases over 50-fold and greater than 90% of the thymidine is immediately phosphorylated. These results imply that there is close cooperativity between fertilization-induced uptake and phosphorylation of thymidine. To gain insight into the structural basis of this apparent cooperativity and to provide a partial localization of the kinases, uptake and phosphorylation were measured in centrifuged eggs, and in centrifuged nucleate and anucleate merogons. Electron micrographs show that in these cells, the inner cytoplasmic contents are stratified according to density and displaced within the egg, whereas the outer cortical region of the cytoplasm remains intact. Uptake and phosphorylation of thymidine are fully stimulated in these eggs and merogons after fertilization, suggesting that both processes are mediated by an intact egg cortex. In support of this suggestion, we report that controlled disruption of the egg cortex prior to fertilization by treatment with cytochalasin B (CB) significantly reduces the rates of uptake and phosphorylation after fertilization. The full stimulation of phosphorylation in nucleate and anucleate merogons eliminates any localization of the catalyzing enzymes (thymidine kinase and thymidylate kinase) in the maternal nucleus and other inner cytoplasmic contents differentially segregated by centrifugation.     

Activation of amino acid uptake at fertilization in the sea urchin egg. Requirement for proton compartmentalization during cytosolic alkalosis

The comparative importance of the release of intracellular ionic calcium, Na+/H+ exchange and cytosolic alkalosis as activator signals was studied on the development of amino acid uptake at fertilization in sea urchin eggs. We show that, once stimulated, the rate of valine uptake is greatly dependent upon intracellular pH. Suppression of the Na+/H+ exchange at the time of activation, by applying ionophore (A23187) in sodium-free artificial sea water (ONaASW), inhibits the development of valine influx. This cannot be restored by a further (30 min later) alkalosis by transferring eggs into sea water. Suppressing the alkalosis in the presence of Na+/H+ exchange at fertilization by simultaneous addition of acid into sea water results in activation of the amino acid carrier which exhibits an increased rate of transport as soon as the eggs are replaced in sea water at pH 8.0. The absence of alkalosis in eggs activated in ONaASW can be counterbalanced either by adding NH4Cl 10 mM or by transfer into ASW at pH 9.0 at activation. Ammonia-treated eggs absorbed amino acid as controls, whereas eggs in sea water at pH 9.0 failed to develop a valine uptake system, suggesting that ammonia can completely replace the effect of Na+/H+ exchange. Furthermore, addition of NH4Cl immediately before fertilization conceals the Na+/H+ exchange but stimulates valine uptake as in controls. These data suggest that: the occurrence of the intracellular calcium increase alone is not sufficient for the develpment of the amino acid transport system; cell alkalinization at fertilization derives from the cytoplasmic membrane-located Na+/H+ exchange and an inward movement of protons into a cortical acidic compartment, which is discussed.     

Thymidine uptake by developing sea urchin embryos

Unfertilized Paracentrotus lividus eggs accumulate very little thymidine. Upon fertilization, however, uptake increases sharply. The pool for thymidine and/or its metabolic products is saturated after 40 min of exposure. Its size is expandable and proportional to the initial concentration of thymidine in the medium. The uptake rate is low shortly after fertilization, increases until 40 min after fertilization and remains constant thereafter. Of the radioactivity taken up in the form of [³H]thymidine during a 30 min exposure beginning at 60 min after fertilization, about 1% is associated with the acid-insoluble fraction and 99% with the acid-soluble fraction.     

Thymidine uptake by Paracentrotus eggs during the first cell cycle after fertilization

[³H]thymidine uptake increased in Paracentrotus eggs from 7 min after fertilization, reaching a plateau from 20 min until the end of the first cell cycle. Increased nucleoside phosphorylation also occurred. 100 μM uridine reduced the thymidine uptake by 90–95%.     

Changes in dopamine uptake and developmental effects of dopamine receptor inactivation in the sea urchin

[³H]-dopamine ([³H]-DA) uptake was measured in the presence or absence of the catecholamine uptake inhibitor nomifensine in both unfertilized and fertilized eggs. Specific [³H]-DA uptake depended on time and [³H]-DA concentration; it was high in unfertilized eggs, declined 20–30 min after fertilization, and rose again during cleavage. Irreversible inactivation of dopamine receptors by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) resulted in a complete loss of sensitivity of egg adenylate cyclase to dopamine stimulation. In fertilized eggs treated with EEDQ for 1 hr, restoration of adenylate cyclase activity sensitive to dopamine stimulation could be observed 4 hr after the end of treatment, thus suggesting the appearance of new dopamine receptors in cleaving eggs. Short-term EEDQ treatment on unfertilized eggs, although not impairing fertilization, resulted in cleavage inhibition; the same treatment carried out soon after fertilization, on the other hand, elicited no effect on development. On the contrary, in embryos subjected to continuous treatment with EEDQ, development was impaired independent of the stage at which the treatment was started. © 1995 Wiley-Liss, Inc.     

Effects of pH on the Fertilization Response of the Medaka Egg

The effects of changing the intra- and extracellular pH on the cortical reaction and sperm penetration into eggs were examined in Oryzias latipes. When eggs were inseminated in a saline adjusted to various pH values with different buffers, fertilization took place normally over a wide range of external pH (pH₀) from about 6 to 10 with a peak at 7 to 8.5. The primary causes of the reduced fertilization were impaired motility or immobility of the spermatozoa in the acidic saline and the swelling of the chorion in the alkaline saline. A rise in the intracellular pH (pH_i) shortly after commencement of the cortical reaction was found by monitoring the color of a pH indicator micro-injected into the cytoplasm. The experimental results obtained by microinjection of various pH buffers (pH 4–12) suggest that the intracellular membrane fusion of the plasma membrane with the cortical alveolar membrane (CABD) is affected by the acidic pH_i while the propagation of the CABD and the intercellular membrane fusion between the plasma membranes of the egg and the spermatozoon are affected by the acidic pH₀.     

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.     

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.     

The relation between the increase in reduced nicotinamide nucleotides and the initiation of DNA synthesis in sea urchin eggs

Previous work has suggested that the activation of the sea urchin egg at fertilization is the result of a transient increase in intracellular free calcium and an increase in intracellular pH. We have investigated the absence of nuclear activation in incompletely activated eggs and have found a correlation between nuclear activation and the levels of total reduced nicotinamide nucleotides (NAD[P]H). Eggs activated with ammonia show a similar correlation: besides its action as a weak base in raising intracelluar pH (which we conclude is insufficient to stimulate or maintain nuclear activation as judged by nuclear envelope breakdown or DNA synthesis), ammonia increases NAD(P)H. This increase is associated with the stimulation of 6-³H-thymidine incorporation into egg DNA. Removing ammonia decreases NAD(P)H, and tritiated thymidine incorporation ceases. We conclude that a critical level of NAD(P)H is essential to nuclear activation and that the increase of NAD(P)H at fertilization must be included with the increase in calcium and pH as a causal agent in development.     

Adaptation of cultured human lymphoblasts to growth in citrulline

DNA synthesis is initiated in unfertilized sea urchin eggs (Strongylocentrotus purpuratus and Lytechinus pictus) by exposing them to NH₄OH-sea water (ordinary sea water titrated to pH 9–9.1 with NH₄OH). The eggs are considered to be unfertilized eggs by visual and electro-biological criteria and because they can later be fertilized and then do give visible and electrobiological fertilization reactions. The incorporation of ³H-thymidine proceeds in rounds, the magnitude increasing in successive rounds. It is also reported that the treatment with NH₄OH activates the uptake of thymidine by the eggs, although the internal thymidine builds up more slowly in unfertilized eggs treated with NH₄OH than it does in fertilized eggs. The magnitude of the incorporation of exogenously supplied labelled thymidine into DNA is lower in the NH₄OH-treated unfertilized eggs than in normal fertilized eggs. This difference is not attributed to differences in the amount of DNA synthesized and the explanation is sought in thymidine uptake and nucleotide pathways.     

Evidence for an acidic compartment in sea urchin eggs (Paracentrotus lividus): role at fertilization

The characteristics of [14C]methylamine accumulation by isolated cortices were measured in eggs from three species of sea urchins: Paracentrotus lividus, Arbacia lixula and Sphaerechinus granularis. In all cases, the results pointed to the existence of an acidic compartment in the cortical zone. In P. lividus eggs, cortical granules did not participate in proton storage which likely took place in pigment granules. [14C]Methylamine accumulation was dramatically reduced by monovalent cation ionophores (monensin and nigericin) and by NH4Cl, but not by valinomycin. ATP depletion only partially affected the isotope uptake. Simultaneous measurements of intracellular pH and of external titratable acidity during ammonia treatment of eggs, indicate that after fertilization, eggs increased their capacity to concentrate hydrogen ions in an intracellular store. Following insemination, cortices from P. lividus eggs exhibited a 3-fold increase in [14C]methylamine accumulation. It is concluded that the egg cortical area contains acidic organelles sequestering hydrogen ions by means of an electrogenic H+ pump, and that this mechanism, enhanced at fertilization, participates in a local alkalinization. The role of such a mechanism is discussed.     

Intracellular pH and the kinetics of Rb+ uptake by yeast non-carrier versus mobile carrier-mediated uptake

The effect of changes in the intracellular pH upon the concentration dependence of the Rb⁺ uptake by yeast is investigated. It is shown, that the uptake of Rb⁺ can be described by a mechanism in which the total concentration of primary binding sites at the outer side of the membrane is independent of the intracellular ligand composition and of the membrane potential, and the influx rate constants depend upon the intracellular pH and/or upon the membrane potential. It is argued that the involvement of a mobile carrier mechanism is not likely.     

Sulfated acid mucopolysaccharides in the cortical granules of eggs. Effects of quaternary ammonium salts on fertilization

Sulfated acid mucopolysaccharides have been shown to be constituents of cortical granules in sea urchin and vertebrate eggs. These observations were made possible by retaining soluble acid mucopolysaccharides in situ within the eggs by precipitation during fixation with cetyltrimethyl-ammonium bromide, a quaternary ammonium salt. The sulfated mucopolysaccharides were then identified by staining with Astrablau at pH 0.2 and also by reaction with sodium rhodizonate. Staining reaction with Alcian blue at pH 2.5 showed that carboxylated mucopolysaccharides may also be present in cortical granules. The natural ionic environment of these eggs would favor the formation of very stable complexes between sulfated mucopolysaccharides and quaternary ammonium salts. Brief exposure of unfertilized sea urchin eggs to several quaternary ammonium compounds produced a residual adverse effect on subsequent fertilization in terms of increased vulnerability to polyspermy and reduced fertilizability. These results suggest that sulfated acid mucopolysaccharides participate in the function of the cortical granules and the establishment of the block to polyspermy at fertilization, and possibly in other cellular secretory processes.     

Thymidine kinase activation in unfertilized sea urchin eggs by homogenization and fertilization

Kinetics of in vivo phosphorylation of ³H-thymidine taken up by sea urchin eggs was compared between unfertilized and fertilized eggs. The percentage of phosphorylated ³H-thymidine in the total acid-soluble radioactivity in the cell increased with increasing incubation time within the first several minutes of incubation in the unfertilized eggs, while nearly 100% of phosphorylation of thymidine was observed without regards to the incubation time and in spite of a tremendous increase in the net uptake of thymidine in the fertilized eggs, suggesting possible activation of thymidine kinase occurring soon after fertilization.In contrast to the in vivo finding, the thymidine kinase activity in unfertilized egg homogenates was found in general to be almost as large as that in fertilized egg homogenates. However, when the enzyme activity was assayed within a short period (30 min) after homogenization of unfertilized eggs, the activity was found to increase more or less with time after homogenization, reaching a level equal to that in fertilized egg homogenates. This enzyme activation after homogenization was especially marked in case of Pseudocentrotus eggs and sometimes amounted to a several fold increase.Preliminary investigations revealed possible involvement of some redox reaction(s) in the thymidine kinase activation during and/or after homogenization of unfertilized sea urchin 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.     

Intracellular pH controls protein synthesis rate in the sea urchine egg and early embryo.

Direct comparisons between intracellular pH and protein synthesis in the sea urchin egg and early embryo show that pH controls protein synthesis rate in a highly sensitive and reversible manner. The entire increase and maintenance of protein synthesis at fertilization or parthenogenetic activation could be accounted for by a permanent increase in intracellular pH. However, unfertilized eggs whose intracellular pH has been raised artificially by ammonia take at least 30 min longer to reach the rate of protein synthesis seen in fertilized eggs. This time lag for ammonia activation and the decrease in protein synthesis rate during mitosis suggest that other unknown factors can also influence protein synthesis rate during fertilization and early embryogenesis.     

Adenosine receptor activation in quiescent Swiss 3T3 cells. Enhancement of cAMP levels, DNA synthesis and cell division

Anti-tubulin immunofluorescence microscopy is used here to demonstrate that eggs of Lytechinus variegatus are induced to assemble cytoplasmic microtubules upon artificial activation. These microtubules progress through three distinct configurations followed by cycles of abortive division. The first of these is a configuration in which microtubules are found in a disordered network near the egg cortex; the progressive thickening of the microtubule-containing layer appears to be responsible for the centripetal movement of the egg nucleus that occurs shortly after activation. These microtubules are replaced at about 40 min by a population of long, radially arrayed microtubules, which are restructured by about 70 min to form the apolar mitotic apparatus. Each of the microtubule configurations characteristic of activated eggs becomes more prominent when eggs are treated at the appropriate times after activation with the microtubule-stabilizing drug taxol. Any microtubule organizing centers within the activated egg must have very limited authority, since aster-like structures are not seen, and microtubules are not observed to be closely associated with the nucleus or egg cortex. Activation of eggs with ammonia in Ca²⁺-free sea water (a treatment that bypasses the cortical reaction and the Ca²⁺ transient) induces the appearance of microtubules as readily and in the same patterns as does treatment with ionophore A23187 or butyric acid, both of which activate by inducing an intracellular calcium release and the cortical reaction.     

Bioelectric responses at fertilization: Separation of the events associated with insemination from those due to the cortical reaction in sea urchin,Lytechinus variegatus

The bioelectric responses at fertilization of the sea urchin Lytechinus variegatus are a complex series of membrane potential and resistance changes that occur concomitant with gamete fusion, ionic fluxes, and the cortical granule discharge. This work attempts to separate the electrical effects of sperm-egg interactions from those of the cortical reactions. Two approaches were taken to discern the electrical events associated with insemination, distinct from cortical granule discharge: (1) fertilization of eggs treated with 3% urethane, 10 mM procaine, or 10 mM nicotine, to prevent the cortical reaction and (2) refertilization of fertilized eggs (denuded with 1 mM aminotriazole containing 1 mg/ml soybean trypsin inhibitor). Cortical granule discharge in the absence of sperm incorporation was investigated by artificial activation with 5 μM A23187 or by fertilization in the presence of 10 μM cytochalasin D, which prevents incorporation. These results are consistent with a model in which the sperm-egg interaction triggers both a rapid (50–400 msec), but minor (?10 mV), electrical transient that leads to an action potential and then both the Na⁺-dependent fast block to polyspermy and the late block resulting from the secretion of the cortical granules.     

Inhibitory Effects of Diltiazem and Verapamil, Calcium Antagonists, on Fertilization-Induced Increase in the Phosphorylase Reaction in Echiuroid Eggs

The calcium antagonists diltiazem and verapamil at 100 μM caused considerable inhibition of the glycolysis system in recently fertilized eggs of the echiuroid, Urechis unicinctus . The levels of glycolytic intermediates in eggs were found to be higher 5 min after insemination than before fertilization while the levels of adenine nucleotides and inorganic phosphate were almost the same before and after fertilization. Addition of diltiazem or verapamil 30 sec after insemination did not inhibit fertilization, but resulted in maintenance of as low levels of glycolytic intermediates as in unfertilized eggs. The apparent mass action ratio in the phosphorylase step, calculated from the levles of glucose-1-phosphate and inorganic phosphate was normally higher in fertilized eggs than in unfertilized eggs, but was maintained at as low a level as in unfertilized eggs by adding these compounds 30 sec after insemination. Phosphorylase a activity also normally increased after insemination, but was maintained at a low level in fertilized eggs by adding these compounds. These compounds also inhibited the increased ⁴⁵Ca²⁺ uptake normally observed after fertilization. These results suggest that after fertilization, the Ca²⁺ level increases associated with fertilization-induced Ca²⁺ influx and that this stimulates Ca²⁺ dependent protein kinase to phosphorylate phosphorylase b , resulting in an increased rate of the phosphorylase reaction.     

Acidic vesicles and the uptake of amines by sea urchin eggs

The radioactive amine [¹⁴C]methylamine is accumulated to a great extent by eggs, with kinetics that are dependent upon temperature (Q₁₀ = 5) and sensitive to metabolic inhibitors. Efflux of [¹⁴C]methylamine from eggs (preloaded with tracer concentrations) is increased immediately after fertilization or NH₄Cl activation. Fluorescent amines (9-aminoacridine (9AA) and acridine orange (AO)) are concentrated in small intracellular compartments, presumably vesicles. The possible role of these vesicles in the accumulation of amines by sea urchin eggs and the activation of the metabolism that ensues is discussed.