Compartmentation of thymidine kinase in unfertilized sea urchin eggs and possible release of thymidine kinase from particulates in activated eggs

The thymidine kinase activity of homogenates of unfertilized eggs of the sea urchin, Hemicentrotus pulcherrimus, in 1 M NaCl was always lower than that of homogenates of the unfertilized eggs in hypotonic media or homogenates of the fertilized or ammonia-activated eggs in 1 M NaCl by 30–50%. Sonication of the unfertilized egg homogenates in 1 M NaCl resulted in the elevation of thymidine kinase activity up to a level in the fertilized or ammonia-activated egg homogenates which is not affected by sonication. Differential centrifugation of unfertilized egg homogenates in 1 M NaCl revealed that the latent thymidine kinase is associated with the 1500g pellet or even with the 200g pellet. Exposure of the 1500g pellet to sonication, hypotonic media, 0.3% Triton X-100 in 1 M NaCl, and 2 M propyleneglycol resulted in the elevation of thymidine kinase, which was eventually shown to be no longer bound to the pellet fraction. Latent thymidine kinase was not detected in the 1500g pellet prepared from the fertilized egg homogenate in 1 M NaCl. These findings seem to suggest that thymidine kinase in unfertilized eggs may be sequestered, at least partly, in some large intracellular structures but may be released from them upon fertilization or ammonia activation, in accordance with our earlier observation on the apparent activation of thymidine kinase afer fertilization.     

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.     

Phospholipid metabolism following fertilization in sea urchin eggs and embryos.

Phospholipid metabolism during early development was examined in the sea urchins Stronglyocentrotus purpuratus and Lytechinus pictus. Transport of ³H-choline was stimulated fivefold following fertilization in both species. However, the actual percent incorporation of labeled precursors into phospholipids from the TCA soluble pool did not change at fertilization. There was a slight increase in transport of ¹⁴C-ethanolamine at fertilization but again there was no change in its percent incorporation into phospholipids. When eggs were preloaded with ³H-choline or ¹⁴C-ethanolamine and fertilized, the eggs or embryos showed similar patterns of incorporation into phospholipids. There was no significant change in the percent phosphorylation of choline in fertilized or unfertilized eggs.An investigation was made of the activity of choline kinase, the first enzyme in the biosynthesis of phosphatidylcholine. This enzyme was found to have similar activities in fertilized and unfertilized eggs using a variety of homogenization media. The activity of choline kinase was found to decrease slightly in activity at fertilization and reach a maximum activity by gastrula.These results indicate that there is no activation of phospholipid synthesis at fertilization of sea urchin eggs. Apparent increased incorporation actually reflects increased transport of precursors and not de novo synthesis.     

Mechanism for Increase in Intracellular Concentration of Free Calcium in Fertilized Sea Urchin Egg : A Method for Estimating Intracellular Concentration of Free Calcium

Intracellular free calcium concentration in the sea urchin egg was calculated to increase from 0.1 mM in an unfertilized egg to 1 mM in a fertilized egg 10 min after fertilization, based on measurement of the dissociation constant between free calcium and sea urchin egg homogenate. The dissociation constant between free calcium (dialyzable calcium) and homogenate of sea urchin eggs was measured by means of dialysis equilibrium. The dissociation constant of the unfertilized egg was about 10^–4 M and that of the fertilized egg was about 10–3 M in three species of sea urchin, Hemicentrotus pulcherrimus, Anthocidaris crassispina, and Pseudocentrotus depressus. An increase in the dissociation constant of the unfertilized egg homogenate was observed after the addition of calcium ion at a concentration above 0.3 mM, the dissociation constant becoming the same as that observed in the fertilized egg homogenate after the administration of CaCl₂ at a concentration above 1 mM. Sodium ion also caused a decrease in the calcium-binding ability of the unfertilized egg homogenate. Therefore, penetration of calcium ion or sodium ion upon fertilization might induce an increase in the dissociation constant and then intracellular concentration of free calcium would increase at fertilization. Almost all calcium-binding ability of the egg homogenate was found in the microsomal fraction, and the substance which bound calcium was thought to be protein in nature, since trypsin could decrease the level of calcium-binding substance in the homogenate of the eggs.     

Prolonged incubation in seawater induces a DNA-dependent protein phosphorylation activity in Arbacia punctulata eggs

Various protein kinases are activated in eggs in response to fertilization. We have previously shown that the induction of DNA-dependent protein phosphorylation activity in the sea urchin eggs is triggered by fertilization. The present study demonstrates that the activation of a DNA-dependent serine/threonine kinase in unfertilized eggs of Arbacia punctulata can be achieved without fertilization. Prolonged incubation in seawater resulted in the activation of the eggs with concomitant induction of DNA-dependent protein phosphorylation activity. The activated eggs when fertilized show a slight increase in the phosphorylation activity 10-min post-insemination. The activity gradually declines as the first and second cleavages proceed. The cytoplasmic extracts of the blastulae, gastrulae, and plutei lack the enzyme activity. These findings reveal that not only fertilization but also egg activation serves as a signal for the induction of a DNA-dependent protein phosphorylation activity in sea urchin eggs suggesting that sperm-entry is not required for the induction of the enzyme activity.     

Altered in vitro phosphorylation of specific proteins accompanies fertilization of Strongylocentrotus purpuratus eggs

Protein phosphorylation in eggs of Strongylocentrotus purpuratus was examined by incubation of egg homogenates with γ[³²P]ATP. Individual phosphorylated proteins were detected by autoradiography after electrophoresis of the disaggregated proteins on SDS-polyacrylamide slab gels. Nearly all of the radioactivity was labile to treatment with pronase, but not to ribonuclease or hydroxylamine, suggesting it to be in the form of protein phosphoesters. The pH dependence for phosphorylation was broad, with cyclic 3′,5′-adenosine monophosphate (cAMP)-dependent phosphorylation optimal at pH 7.7. Phosphorylation of several protein species at pH 7.7 was altered in homogenates of fertilized eggs, when compared to that of unfertilized eggs. These relative increase or decreases in intensity detected by autoradiography were not accompanied by corresponding changes in protein staining of the gels, suggesting that the differences were not due to major shifts in overall protein composition. Most of the alterations in phosphorylation were evident in homogenates made within 5 min after fertilization and were stable until the first cell division. The alterations were also found with homogenates of eggs activated with the divalent ionophore A23187, and some, but not all were present following treatment with ammonia, under conditions that induce a partial metabolic activation of eggs. The results suggest that fertilization promotes alterations in the availability of phosphorylation sites in egg homogenates, or changes in the activity of the egg kinases toward specific protein substrates, that may play a role in the activation of egg metabolism.     

Activation of phosphorylase in sea urchin eggs by Ca and cyclic 3′5′-AMP : A possible mechanism of the regulation of its activity at fertilization

γ-Glucan phosphorylase (EC 2.4.1.1) activity in homogenates of unfertilized and fertilized sea urchin eggs, Pseudocentrotus depressus and Hemicentrotus pulcherrimus, has been studied.The phosphorylase exhibits a pH optimum at 6.4 and occurs in two forms, AMP-independent and AMP-dependent, the latter showing maximum activity in the presence of 10 mM AMP.By as little as 5 min after insemination a significant increase in the total phosphorylase activity of the egg as well as in the AMP-independent form is demonstrable. The highest specific enzyme activity is consistently found in the supernatant fraction of both the fertilized and the unfertilized egg homogenate. Thus, fertilization does not appear to cause activation of the enzyme by stimulating a change from a particulate-bound to a soluble state.The phosphorylase activity was compared after incubation of homogenates with a variety of agents potentially able to alter the enzyme activity. Combination of suitable amount of cyclic 3′5′-AMP (cAMP) and Ca²⁺ showed the maximal activating effect on the AMP-independent form of phosphorylase. The fertilization-induced increase of Ca²⁺ and of cAMP were discussed as possible activators of phosphorylase, and consequently, of carbohydrate metabolism.     

DNA sequences flanking the starts of the hsp 70 and alpha beta heat shock genes are homologous

To determine whether ribosomes have a role in the postfertilization activation of protein synthesis in sea urchin eggs, we measured the translational activity of ribosomes isolated from unfertilized eggs and embryos of Strongylocentrotus purpuratus. Numerous previous studies have indicated few if any differences in the activity of such ribosomes. However, by using improved physiological isolation and in vitro conditions, we have found important differences in the activities of egg and embryo ribosomes. Ribosomes obtained from blastula polyribosomes were active in translating reticulocyte mRNA in a ribosome-dependent cell-free translation system, whereas ribosomes obtained from unfertilized eggs became fully active only after a characteristic, reproducible delay of up to 15 min at 26°C. The extent of this delay varied with incubation pH, but not with concentrations of K⁺, Mg²⁺, initiation factors, or mRNA. However, at incubation pH between 6.90 and 7.65, the egg ribosomes were always less active than blastula ribosomes.     

Regulation of tyrosine-specific kinase activity at fertilization

The sea urchin egg contains a protein kinase which phosphorylates tyrosine residues of endogenous membrane proteins as well as synthetic peptide substrates. Fertilization results in an increase in tyrosine kinase activity which first becomes apparent 20–30 min postinsemination and continues throughout the early cleavage stages. This effect can be duplicated by treating unfertilized eggs with the calcium ionophore A23187. The kinase activity begins to increase about 20 min after addition of the ionophore and continues to increase for at least 1 hr. Both the time course and the extent of kinase activity in ionophore treated eggs closely resemble the effects of fertilization. The concentration of ionophore necessary to induce the increase in enzyme activity (2–5 μM) is also effective in inducing the cortical reaction. Neither A23187 nor calcium has a significant effect on the kinase activity of egg homogenates solubilized in NP40, suggesting that the ionophore affects tyrosine phosphorylation indirectly, possibly acting through other calcium-sensitive enzymes.     

Calcium-mediated inactivation of the MAP kinase pathway in sea urchin eggs at fertilization.

We have evaluated the regulation of a 43-kDa MAP kinase in sea urchin eggs. Both MAP kinase and MEK (MAP kinase kinase) are phosphorylated and active in unfertilized eggs while both are dephosphorylated and inactivated after fertilization, although with distinct kinetics. Reactivation of MEK or the 43-kDa MAP kinase prior to or during the first cell division was not detected. Confocal immunolocalization microscopy revealed that phosphorylated (active) MAP kinase is present primarily in the nucleus of the unfertilized egg, with some of the phosphorylated form in the cytoplasm as well. Incubation of unfertilized eggs in the MEK inhibitor U0126 (0.5 microM) resulted in the inactivation of MEK and MAP kinase within 30 min. Incubation in low concentrations of U0126 (sufficient to inactivate MEK and MAP kinase) after fertilization had no effect on progression through the embryonic cell cycle. Microinjection of active mammalian MAP kinase phosphatase (MKP-3) resulted in inactivation of MAP kinase in unfertilized eggs, as did addition of MKP-3 to lysates of unfertilized eggs. Incubation of unfertilized eggs in the Ca(2+) ionophore A23187 led to inactivation of MEK and MAP kinase with the same kinetics as observed with sperm-induced egg activation. This suggests that calcium may be deactivating MEK and/or activating a MAP kinase-directed phosphatase. A cell-free system was used to evaluate the activation of phosphatase separately from MEK inactivation. Unfertilized egg lysates were treated with U0126 to inactivate MEK and then Ca(2+) was added. This resulted in increased MAP kinase phosphatase activity. Therefore, MAP kinase inactivation at fertilization in sea urchin eggs likely is the result of a combination of MEK inactivation and phosphatase activation that are directly or indirectly responsive to Ca(2+).     

Heat labile activating substance for ornithine decarboxylase activity in sea urchin eggs

In sea urchin eggs, the activity of ornithine decarboxylase (ODC) [ E C 4.1.1.17] is detectable only in the particulate fraction yielded by centrifuging egg homogenates at 10,000g for 30 minutes. ODC activity in the particulate fraction isolated from fertilized eggs is higher than that from unferti-lized eggs. ODC activity in the particulate fraction isolated from either unfertilized or fertilized eggs is enhanced by adding the supernatant fraction obtained by centrifugation at 105,000g for two hours. Heating this supernatant at 70°C for 15 minutes results In complete loss of the stimulating capacity for ODC activity. Sea urchin eggs seem to contain heat labile activating substance(s) for ODC activity. The substance does not pass through the ultrafiltration membrane Diafro UM–10. Only eggs and unhatched embryos, in which mitosis occurs frequently, contain the activating substance. In the presence of the activating substance, Ca²⁺enhanced ODC activity.     

Cytoplasmic and sperm nuclear transformations in fertilized ammonia-activated sea urchin (Arbacia punctulata) eggs

Studies examining cytoplasmic and sperm nuclear transformations in sea urchin (Arbacia punctulata) eggs inseminated at different periods after ammonia activation have been caried out at the light- and electron-microscopic levels of observation. Arbaca eggs treated with ammonia-seawater demonstrated chromosome condensation after DNA synthesis and underwent a chromosome cycle similar to that described for Lytechinus [Mazia, 1947]. Cortical granule reaction, fertilization cone formation, and sperm aster development in eggs fertilized at 20 (interphase), 50 (prometaphase), and 180 (interphase) min after ammonia activation were structurally simialr to processes in untreated zygotes. Cyclical changes in the formation of fertilization cones and sperm asters, as reported for eggs fertilized after activation by agents that induce a cortical granule reaction, were not observed. Although sperm nuclear transformations were prolonged (14 vs 18 min), male pronuclei that developed in eggs fertilized 20 min after ammonia activation were morphologically similar to those observed in fertilized, untreated ova and incorporated ³H-thymidine. Sperm incorporated into eggs at 50 min after ammonia activation underwent nuclear envelope breakdown and chromatin despersion; however, ³H-thymidine incorporation was not observed, and male pronuclei rarely developed (less than 5% of all specimens examined). Subsequent to dispersion, the paternal chromatin condensed into chromosomes which were associated with an aster. These results demonstrate that although ammonia-activated eggs inseminated at interphase or prometaphase undergo similar cytoplasmic alterations, sperm nuclear transformations vary with the chromosome cycle of the egg.     

CHANGE IN THE ACTIVITY OF PYRUVATE DEHYDROGENASE COMPLEX IN SEA URCHIN EGGS FOLLOWING FERTILIZATION*

The activity of the pyruvate dehydrogenase complex in sea urchin eggs is localized in the crude mitochondrial fraction. The activity of the enzyme complex in the intact mitochondrial fraction of unfertilized eggs is too low to be estimated and is enhanced upon fertilization with a 5-min lag period. The activity of the enzyme complex in unfertilized eggs is enhanced by Ca²⁺at concentrations between 5 × 10^?5 M and 10^?3 M. The activity in fertilized eggs is blocked after incubation with 2 mM ATP, and the block of the activity is also released by Ca²⁺. The blockage of the enzyme complex activity is accompanied by phosphorylation of proteins, and release of the block by Ca²⁺ is concomitantly followed by the dephosphorylation of proteins in the mitochondrial fraction. The enzyme complex in unfertilized eggs will be assumed to be the one inhibited by phosphorylation. The enzyme complex will be activated upon fertilization as a consequence of the dephosphorylation, that is caused by the increase in intracellular concentration of Ca²⁺.     

Hexokinase activity from eggs of the sea urchin Arbacia punctulata

1. The hexokinase activity of homogenates of eggs and embryos of the sea urchin Arbacia punctulata has been measured. Expressed as micrograms glucose consumed at 20°C., per hour per milligram of protein the following values were obtained: unfertilized eggs, 67; fertilized eggs, 72; 24 hour plutei, 94; 48 hour plutei, 226. The concentration of the enzyme in the eggs is small and may be calculated to be about 0.001 per cent of the dry weight of unfertilized eggs. 2. The hexokinase activity of the egg homogenate was virtually all recovered in the supernatant fraction when the homogenate was centrifuged at 20,000 x g for 30 minutes and was found to have the following properties: The concentrations for half maximal hexokinase activity with various substrates were, approximately: Glucose, 0,00003 M; fructose, 0.00075; mannose, 0.00007; 2-desoxyglucose, 0.00025. The relative rates of phosphorylation of various sugars by the supernate fraction when saturated with substrate were, approximately: Glucose, 1.0; mannose, 1.2; fructose, 1.8; 2-desoxyglucose, 2.0; glucosamine, 0.6. Adenosinediphosphate and glucose-6-phosphate inhibited the enzyme. No evidence for more than one hexokinase in the Arbacia extracts was found.     

Intracellular pH change does not accompany egg activation in the mouse

In the sea urchin, some other marine invertebrates, and the frog, Xenopus, egg activation at fertilization is accompanied by an increase in intracellular pH (pH_i). We measured pH_i, in germinal vesicle (GV)-intact mouse oocytes, ovulated eggs, and in vivo fertilized zygotes using the pH indicator dye, SNARF-1. The mean pH_i was 6.96 ± 0.004 (± SEM) in GV-intact oocytes, 7.00 ± 0.01 in ovulated, unfertilized eggs, and 7.02 ± 0.01 in fertilized zygotes, indicating no sustained changes in pH_i after germinal vesicle breakdown (GVBD) or fertilization. To examine whether transient changes in pH_i occur shortly after egg activation, mouse eggs were parthenogenetically activated by 7% ethanol in phosphate buffered saline (PBS); no significant change in pH_i followed ethanol activation. Since increased Na⁺/H⁺ antiporter activity is responsible for pH_i increase in the sea urchin, pH_i was measured in the absence of added bicarbonate or CO₂ la condition under which the antiporter would be the only major pH_i regulatory mechanism able to operate, since the others were bicarbonate- dependent) in GV-intact oocytes, ovulated eggs, and in vivo fertilized zygotes to determine whether a Na⁺/H⁺ antiporter was activated. There was no physiologically significant difference in pH_i after GVBD or fertilization, when pH_i was measured in bicarbonate-free medium, nor any change upon parthenogenetic activation. Thus, a change in pH_i is not a feature of egg activation in the mouse. © 1996 Wiley-Liss, Inc.     

The cytolytic isolation of the cortex of the sea urchin egg

When the vitelline layer of sea urchin eggs (Lytechinus pictus) is disrupted by trypsin or dithiothreitol and the eggs are placed in an isosmotic medium devoid of Ca²⁺, cytolysis of the eggs occurs. During lysis the entire egg cortex peels off in one piece. Lysis is temperature and pH dependent and is inhibited by cytochalasin B. Cortices from unfertilized eggs contain seven major macromolecular components. A 42K-dalton component is believed to be actin, representing between 12 and 27% of the total protein. Cortices from fertilized eggs may contain between 50 and 65% actin. The actin appears to increase the strength of its attachment to the cortex after fertilization. This method of isolating the entire cortex may be useful for studying structural and enzymatic changes which may occur in the cortex during the cell cycle.     

Protein tyrosine kinase-dependent release of intracellular calcium in the sea urchin egg

The aminoguanide, methylglyoxal bis(guanylhydrazone) (MGBG), was shown to stimulate phosphorylation of RR-SRC, a synthetic protein tyrosine kinase (PTK) substrate, and different levels of tyrosyl phosphorylation of endogenous proteins in a sea urchin egg membrane-cortex preparation. Stimulating protein tyrosine kinase activity in the sea urchin egg stimulated intracellular Ca2+ release, because microinjection of 1-5 mM of MGBG into unfertilized eggs triggered a transient rise in intracellular Ca2+ activity ([Ca2+]i) after a brief latent period. Pretreating eggs with PTK-specific inhibitors, genistein or tyrphostin B42, significantly inhibited the MGBG-induced rise in [Ca2+]i. Methylglyoxal bis(guanylhydrazone) stimulation of PTK activities in the unfertilized sea urchin egg appeared to trigger Ca2+ release through phospholipase C (PLC)-dependent inositol 1,4,5-trisphosphate (InsP3) production. The MGBG-induced Ca2+ response could be suppressed in eggs preloaded with the InsP3 receptor antagonist, heparin, and was reduced in eggs pretreated with U73122, a PLC inhibitor. However, the response was unchanged in eggs treated with nicotinamide, an inhibitor of ADP-ribosyl cyclase, or nifedipine, an inhibitor of nicotinic acid adenine dinucleotide phosphate activity. These results suggest that MGBG may be useful as a chemical agonist of PTK in sea urchin eggs and allow direct testing of the PTK requirement for the transient rise in [Ca2+]i in sea urchin eggs during fertilization. Although genistein was observed to significantly delay the onset, the sperm-induced Ca2+ response in PTK inhibitor-loaded eggs otherwise appeared normal. Therefore, it was concluded that sea urchin eggs contain a PTK-dependent pathway that can mediate intracellular Ca2+ release, but PTK activity does not appear to be required for the fertilization response.     

Uptake of deuterium into proteins of fertilized and unfertilized Arbacia eggs suspended in heavy water

When fertilized and unfertilized eggs of Arbacia punctulata are suspended in heavy water, deuterium is incorporated into stable positions in the egg proteins. The rate of incorporation of the isotope is considerably greater in fertilized than in unfertilized eggs, and is accelerated at the time of formation of the blastula. The result of calculation of the maximum deuterium concentration which would be reached on complete turnover indicates that at least one out of every ten stably bound hydrogen atoms of the egg proteins is a deuterium atom. This has been interpreted as evidence that at the time of formation of the sea urchin blastula and in the period of development which follows, synthesis and breakdown are simultaneous processes leading to the redistribution of amino acids among the egg proteins.     

An assessment of the masked message hypothesis: sea urchin egg messenger ribonucleoprotein complexes are efficient templates for in vitro protein synthesis

The rate of protein synthesis in unfertilized sea urchin eggs is very low, although all components for protein synthesis are present. To determine whether egg messenger RNAs are unavailable for translation because of “masking” by phenol-soluble inhibitors, crude and purified nonpolyribosomal messenger ribonucleoprotein complexes (mRNPs) from eggs of Strongylocentrotus purpuratus were translated in vitro in a wheat germ cell-free system. Crude and purified egg mRNPs were nearly as translatable as the mRNAs extracted from the mRNPs, suggesting that the mRNPs were not masked. No difference in the relative translational activities of mRNPs and their constituent mRNAs was revealed by isolating the mRNPs in buffers of different ionic strength or in the presence of protease and ribonuclease inhibitors. Furthermore, kinetic analysis of the in vitro translation and translation of the mRNPs and mRNAs at several concentrations of K⁺, Mg²⁺, and template all indicate that mRNPs are efficient templates for directing protein synthesis. Separation on polyacrylamide gels of the products of in vitro and in vivo translation demonstrated that both mRNPs and mRNAs extracted from the mRNPs synthesized in vitro high-molecular-weight polypeptides, some of which were also synthesized in vivo. Although sea urchin egg mRNPs may not be masked, there are several alternative mechanisms for regulating translation in the egg.     

An ultrastructural immunocytochemical localization of hyalin in the sea urchin egg

The fertilized sea urchin egg is invested by the hyaline layer, a thick extracellular coat which is necessary for normal development. On the basis of ultrastructural studies and the fact that hyalin is released during the time of the cortical reaction, it has been generally accepted that hyalin is derived from the cortical granules. However, this has never been proven definitely, and recently, it has been reported that hyalin is a membrane and/or cell surface protein. To determine where hyalin is stored, we carried out an ultrastructural immunocytochemical localization of hyalin in the unfertilized egg. Hyalin purified from isolated hyaline layers was used to immunize rabbits. Antisera so obtained were shown to be hyalin specific following absorption with a combination of sea urchin proteins. Immunocytochemical localizations were carried out on sections of Epon-embedded material using protein A-coated gold particles as an antibody marker. Our results demonstrate that, prior to fertilization, hyalin is stored in the homogeneous component of the cortical granule in Strongylocentrotus droebachiensis and Strongylocentrotus purpuratus. Labeling of small cortical vesicles in both unfertilized and fertilized eggs, suggests that these vesicles may contain a secondary reservoir of hyalin.