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.     

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.     

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.     

Effect of diesel fuel hydrocarbons on embryogenesis and 45Ca2+ uptake by unfertilized eggs of sea urchin,Strongylocentrotus intermedius

1. The quality of unfertilized eggs of the sea urchin Strongylocentrotus intermedius, kept for a long time (50 days) in a sea water containing water soluble hydrocarbons of diesel fuel in sublethal concentrations (0.3–0.04 mg/l), was assessed through observation of embryogenesis and the intensity of ⁴⁵Ca²⁺ uptake.2. It has been shown that such treatment led to delay and asynchronism of embryonal and larval development and to appearance of a greater number of abnormalities compared to the control.3. Unfertilized eggs of sea urchins exposed to the hydrocarbons in sublethal concentrations accumu- lated 30–60% more ⁴⁵Ca²⁺ than those of control animals. Short-term incubation (2 hr) of eggs at the same hydrocarbon concentrations did not change ⁴⁵Ca²⁺ uptake by unfertilized eggs of control animals.4. The increase of hydrocarbon concentration up to 1 mg/l (i.e. to a concentration causing disturbance of embryogenesis in acute experiments) in short-term experiments caused a small elevation in the ⁴⁵Ca²⁺ uptake by unfertilized eggs of control animals (30% more than in untreated eggs).5. Ionomycin-induced (concentration 10⁻⁸−10⁻⁹) increase of ⁴⁵Ca²⁺ uptake by unfertilized eggs (50–100% more than the untreated eggs) caused the same disturbance of embryogenesis as under hydrocarbon exposure.6. It is suggested that one of the mechanisms inducing the deleterious effect of hydrocarbons in sea urchin gametes is related to the increase of membrane permeability to calcium ions.     

DNA synthesis in unfertilized sea urchin eggs can be turned on and turned off by the addition and removal of procaine hydrochloride.

Unfertilized sea urchin eggs turn on thymidine transport, DNA synthesis and the chromosome cycle in response to procaine hydrochloride. The rates to which these processes activate depend on the extracellular concentration of procaine. Removing procaine turns off DNA synthesis and the chromosome cycle, re-adding procaine turns these processes on once more. Thymidine transport does not turn off after procaine removal. It remains on at the rate it had reached at the time of procaine removal. During a 12-hr period, unfertilized eggs in procaine complete four S-phases, while normal, fertilized embryos complete eight. Tritiated thymidine incorporation into DNA is very low in procaine-treated eggs. This is because in procaine the thymidine transport system is relatively inactive compared to that of fertilized eggs. The data suggest eggs may possess homeostatic mechanisms that actively suppress the DNA synthetic pathway and the chromosome cycle. This system for turning on and off DNA synthesis may prove useful in further analyses of the factors controlling DNA synthesis during early development.     

Effects of stubble and N fertilization management on N availability and uptake under successive rice (Oryza sativa L.) crops

Experiments were conducted in fields which had a history of nil to four rice (Oryza sativa L.) crops during the previous four summers. Incorporating stubble after each harvest reduced soil nitrate-N content between crops, but increased soil N mineralization potential. During the fourth successive crop, plots where stubble had been incorporated after the previous three harvests had an average 21% more soil NH₄N and 22% more N uptake than plots where stubble had been burnt.Soil fertility fell rapidly with increasing numbers of crops, and the unfertilized fifth crop accumulated approximately half the N (60 kg N ha^-1) found in the unfertilized first crop (116 kg). Fertilizer N alleviated the effects of annual cropping; the application of 210 kg N ha^-1 to the fifth crop (uptake of 156 kg N ha^-1) resulted in similar N uptake to the first crop fertilized with 50 kg N ha^-1 (154 kg N ha^-1).Applying N at sowing had no significant effect on soil NH₄-N concentration after permanent flood (PF), while N application at PF resulted in increased NH₄-N concentration and N uptake until panicle initiation (PI). N applied at PI increased soil NH₄-N concentration at least until the microspore stage.Management factors such as stubble incorporation and increasing N application rate, maintained N supply and enabled successive rice crops to accumulate similar quantities of N at maturity.     

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.     

Increased uptake of thymidine in the activation of sea urchin eggs : Specificity of uptake and dependence on internal pH, the cortical reaction, and external sodium

The uptake of thymidine in sea urchin eggs is considered in terms of its specificity, the cortical reaction, and the increase of intracellular pH following fertilization. The rate of uptake increases greater than 50-fold after fertilization. All deoxyribonucleosides and ribonucleosides tested compete with thymidine for transport sites. Free pyrimidine and purine bases, deoxyribonucleotides, and amino acids do not compete, showing that the specificity of this uptake lies at the nucleoside level. Uptake may be turned on in unfertilized eggs by treatment with ammonia, a treatment known to by-pass the cortical reaction and raise intracellular pH. However, when compared with uptake in fertilized eggs, it proceeds later and at a lower rate. Both of these deficiencies are overcome by fertilizing the ammonia-treated eggs or treating them with butyric acid or ionophore A23187. These treatments induce the cortical reaction and stimulate an immediate and complete turn-on of thymidine uptake. Superseding these apparent involvements of the cortical reaction and mtracellular pH in thymidine uptake is an extremely strict requirement for extracellular Na⁺.     

CHANGE IN THE ACTIVITY OF LIPASE IN SEA URCHIN EGGS AND EMBRYOS DURING EARLY DEVELOPMENT*

During the early development of the sea urchin, Anthocidaris crassispina, the activity of lipase was maintained at the same level as in unfertilized eggs until the mesenchymal blastula stage (20 hr culture at 20°C) and then increased gradually after gastrulation. The activity in the embryos kept in SO^2?₄-free artificial sea water changed in a similar manner to that in those kept in normal sea water, during the development until 36 hr of fertilization. At 48 hr, the activity in the embryos, which had developed to the permanent blastulae in SO^2?₄-free sea water, was markedly lower than in normal plutei and was similar to that in unfertilized eggs. The lipase activity in fertilized eggs 30 min after fertilization, which was almost the same as that in unfertilized eggs was found mainly to be localized in the precipitate fraction obtained by the centrifugation at 12,000 x g for 20 min, whereas the activity in unfertilized eggs was found in the precipitate by the centrifugation at 105,000 x g for 60 min. Ca²⁺, adenosine 3′, 5′-cyclic monophosphate (cAMP) and guanosine 3′, 5′-cyclic monophosphate (cGMP) had no effect on the lipase activity.     

Sodium-potassium exchange in sea urchin egg. I. Kinetic and biochemical characterization at fertilization

Biochemical and kinetic characteristics of the Na⁺-K⁺ exchange were studied in Paracentrotus lividus eggs. Measurement of the ⁸⁶Rb uptake shows that ouabain-sensitive ⁸⁶Rb uptake is dramatically stimulated within the first minute following fertilization. The Na⁺-K⁺ pump-mediated K⁺ entry presents a maximal rate at 8 min postfertilization and then decreases to reach a plateau within 30 min. We assess that the steep rise in cell K⁺ occurring at fertilization (J.P. Girard, P. Payan, C. Sardet, Exp. Cell. Res. 142:215–221, 1982) does not originate from a net entry of external K⁺. Measured 30 min postfertilization, the half-maximal activation by K⁺ of the ouabain-sensitive Na⁺-K⁺ exchange is 5–6 mM and the ouabain lC₅₀ is 5.10^?5 M. Egg cortices from unfertilized and fertilized eggs show comparable Na⁺-K⁺ ATPase activity with a 50% ouabain-sensitive fraction. V_m and K_m for Na⁺ and K⁺ of the enzyme are of the same order of magnitude in cortices of unfertilized and fertilized eggs. Cortical Na⁺-K⁺ ATPase from unfertilized eggs shows a ten fold increase of activity between pH 6.7 and pH 7.7. The results strongly suggest that the plasma membrane of unfertilized eggs contains a preexisting Na⁺-K⁺ transporting system which is obligatorily stimulated at fertilization.     

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.     

Stimulation of Unfertilized Eggs of the Echiuroid, Urechis unicinctus by Polyamines

Unfertilized eggs of the echiuroid, Urechis unicinctus , were activated by polyamines, such as putrescine, spermidine and spermine at concentrations above 10 μM. Fertilization membrane elevated and germinal vesicle disappeared in unfertilized eggs kept for several min in sea water containing these polyamines. Following the addition of these polyamines, a decrease of pH value in the egg suspension, occurred in a similar manner as observed following fertilization. Several sec after the addition of polyamines to the egg suspension, the respiratoy rate increased very slightly and the sensitivity of the respiration to 2, 4-dinitrophenol, which was lower in unfertilized eggs than in fertilized eggs, became as high as in fertilized ones. Irregular cleavage occurred in the eggs stimulated by polyamines. The incorporation of [³H]-deoxyadenosine into DNA was initiated by adding polyamines in the unfertilized eggs preloaded with the isotope. The rate of [³H]-leucine incorporation into protein in the preloaded unfertilized eggs was also enhanced by polyamines, in almost the same manner as observed following fertilization.     

Changes in the potassium content of sea urchin eggs on fertilization

In the eggs of Arbacia lixula and Paracentrotus lividus an uptake of K occurs during the first 10 minutes following fertilization. Between 10 and 40 minutes K is then released. Both in Arbacia and in Paracentrotus the minimum point of the curve coincides with the nuclear streak stage. A maximum loss of 25 per cent in Arbacia and 20 per cent in Paracentrotus with respect to the amount present in the unfertilized eggs has been found. From 40 minutes up to 1 hour K undergoes a further increase and when the first cleavage sets in the same amount of K is present as in the unfertilized eggs. By treating the eggs with K-free artificial sea water it has been established that about 60 per cent of the K content of the eggs is in a non-diffusible condition. Also under such conditions the eggs when fertilized are able to take up even the very small amount of K present in the medium that was released by them prior to fertilization.     

Uptake of valine and alanine by a neutral amino-acid carrier in sea urchin eggs: cyclic variations in the early cleavage stage

Summary The characteristics of valine and alanine uptake (respectively, preferential substrates of the L and A or ASC systems in mammalian cells) were studied in sea urchin eggs before and 40 min after fertilization. Substrate concentration dependence showed that in unfertilized eggs alanine is absorbed linearly up to an external concentration of 20mm, whereas valine uptake presented a saturable kinetic with aK _m of 6 m. Competition experiments showed that valine is absorbed by a carriermediated transport resembling the L system. Fertilization develops a new Na-dependent system, resembling the ASC system which is specific for neutral amino acids but does not discriminate between them. This system is superimposed on that of the unfertilized egg. In fertilized eggs, amino-acid transport displayed cyclic variations which have been previously associated with cell cleavage. We have found that eggs prevented from cleavage by treatment with antimitotic undergo a sequence of periodic amino-acid uptake timed with the mitotic cycle of untreated eggs. In addition, artificially activated eggs (A23187) which failed to divide showed a time course of amino-acid uptake similar to that observed in fertilized eggs. Furthermore, these variations are independent of protein synthesis. These results suggest to us that a biological clock exists in the cytoplasm or cortex of sea urchin eggs, which may be involved in timing the cell cycle.     

Replication of mitochondrial DNA in the early development of Misgurnus fossilis

Mitochondria isolated from Misgurnus fossilis embryos at various developmental stages were incubated with ³H-dTTP in vitro and the incorporation into mtDNA was determined. It has been found that the rate of mtDNA labeling increases exponentially with a doubling time of 7 hr from 0.01 pmole of ³H-dTMP/mg protein/hr in mitochondria from unfertilized eggs to 0.4 pmoles of ³H-dTMP/mg protein/hr in mitochondria of 35 hr embryos. The pool of intramitochondrial dTTP decreases 2.5 times during the first 10 hr after fertilization, then remains practically constant up to 35 hr of development. The rate of exogenous ³H-dTTP incorporation into the acid soluble pool of isolated mitochondria at two stages is approximately proportional to the pool size. Thus identical specific activities of ³H-dTTP inside mitochondria would be obtained even with pools of different sizes. We conclude that the increase of ³H-dTMP incorporation into mtDNA in development reflects genuine activation of mtDNA synthesis. As early as 6 hr after fertilization the bulk of the label incorporated into mtDNA is found in the fraction associated with covalently closed molecules. This pattern of labeling characteristic for replicating mtDNA is maintained throughout early development. In contrast such preferential label incorporation into the closed circular fraction was not found with mitochondria of unfertilized eggs. Closed mtDNA from unfertilized eggs contains not more than 1% of molecules with D-loops. In 35 hr embryos the corresponding value is equal to about 4%. Activation of mtDNA replication in embryogenesis is probably due to the activation of mechanisms responsible for the generation of primers for replication. DNA polymerase activity solubilized from mitochondria remains unchanged in the course of embryogenesis.     

Comparison of Ca uptake characteristics of microsomal fractions isolated from unfertilized and fertilized sea urchin eggs

The microsomal fraction isolated from sea urchin H. pulcherrimus eggs has the ability to actively accumulate Ca²⁺ in the presence of ATP. The Ca²⁺ uptake was sustained by addition of oxalate and was apparently insensitive to sodium azide. The sequestered microsomal Ca was readily released by the divalent cation ionophore A23187. The microsomal fraction obtained from fertilized eggs accumulated Ca²⁺ about five times more quickly than did that from unfertilized eggs. The increased Ca²⁺ uptake by microsomal fraction obtained from fertilized eggs was due to an increase in the maximum velocity of Ca²⁺ uptake and there was no difference in K_m for calcium between the two fractions.     

Changes of uridine permeability during the maturation of tunicate eggs.

The time course of uridine uptake by eggs and embryos of the tunicate Ascidia callosa was studied using 5-min pulses of [³H]uridine at intervals from the unfertilized egg to the 16-cell embryo. The unfertilized egg is permeable to uridine, but 5 min after fertilization uptake begins to drop, reaching a minimum of 30% of the unfertilized rate about 30 min after fertilization. At 45 min after fertilization, permeability begins to increase, reaching a plateau about 3 hr after fertilization at the two-cell stage. The initial decrease in permeability occurs at first polar body production; the increase at 45 min is coincident with the formation of the second polar body. Substrate concentration experiments up to 200 μM show strict concentration dependence for uridine uptake. The inhibitors p-chloromercuribenzoate (PCMB), dinitrophenol (DNP), and thymidine have little, if any effect on permeability. Cold (?1°C) and Na⁺-free sea water inhibit uptake 60% during all three developmental stages. The changes in permeability may be indicative of temporary reorganization of the plasma membrane during the fertilization-initiated completion of meiosis.     

THE ACCUMULATION OF ELECTROLYTES : III. BEHAVIOR OF SODIUM,POTASSIUM, AND AMMONIUM IN VALONIA

When 0.001 M NH₄Cl is added to sea water containing Valonia macrophysa there seems to be a rapid penetration of undissociated NH₃ (or NH₄OH) which raises the pH value of the sap so that the thermodynamic potential of KOH becomes greater inside than outside and in consequence K leaves the cell: NaOH continues to go in because its thermodynamic potential is greater outside than inside. NH₄Cl accumulates, reaching a much higher concentration inside than outside. This might be explained on the ground that NH₃, after entering, combines with a weak organic acid produced in the cell whose anion is exchanged for the Cl^- of the sea water, or (more probably) the organic acid is exchanged for HCl.     

INHIBITION OF DIHYDROFOLATE REDUCTASE BY PALMITOYL-CoA AND THE REVERSAL OF THE INHIBITION BY SPERMINE AND SPERMIDINE IN THE EGGS OF THE SEA URCHIN,HEMICENTROTUS PULCHERRIMUS*

Dihydrofolate reductase activity in fertilized eggs of the sea urchin, Hemicentrotus pulcherrimus, was almost the same as in unfertilized eggs. Aminopterin inhibited the enzyme competitively with dihydrofolate (FH₂). The apparent K_m value for FH₂ in the dihydrofolate reductase reaction was about 0.1 μM in the crude homogenate of both unfertilized and fertilized eggs. Dihydrofolate reductase in the eggs was also inhibited by palmitoyl-CoA. The inhibition was canceled by polyamines, especially by spermine, but putrescine failed to prevent the enzyme from the inhibition. The change in long-chain acyl-CoA and polyamine concentrations during fertilization are discussed as possible regulatory factors of the enzyme.