THE EFFECT OF PENICILLIN ON EGGS OF THE SEA URCHIN,ARBACIA PUNCTULATA

1. Penicillin in the range of concentration from 250 U/ml. to approximately 2650 U/ml. inhibits the rate of cell division of the fertilized sea urchin egg from 0 to 100 per cent. 2. Penicillin in the same range of concentrations has no effect on the oxygen consumption of the unfertilized or the fertilized eggs. 3. Penicillin is bound by some component of the sea urchin egg in amounts sufficiently large to lower the initial concentration, this binding apparently not being related to the inhibitory action.     

Glucose-6-phosphate and 6-phosphogluconate dehydrogenases from eggs of the sea urchin,Arbacia punctulata

1. Glucose-6-phosphate and 6-phosphogluconate dehydrogenases have been found in homogenates of Arbacia eggs; 95 per cent of the activity toward each substrate is recovered in the supernatant fraction after centrifuging at 20,000 g for 30 minutes. 2. With glucose-6-phosphate as substrate) the rate of TPN reduction by the supernatant fraction from 1 gm. wet weight unfertilized or fertilized eggs was 1.8 to 3.0 micromoles per minute; this rate is sufficient to support a rate of oxygen consumption 24 times that observed for unfertilized, and 6 times that for fertilized, eggs. Pentose was formed from glucose-6-phosphate at a rate 0.3 to 0.5 that of TPN reduction, when both rates were expressed as micromoles per minute. 3. The concentrations of glucose-6-phosphate and 6-phosphogluconate for half maximal activity were each approximately 0.00004 M for the respective enzymes in the supernatant fraction. Maximal activity toward 6-phosphogluconate was 50 to 60 per cent of that toward glucose-6-phosphate. Glucose-6-phosphate dehydrogenase activity was 50 per cent inhibited in presence of 0.00006 M 2,4,5-trichlorophenol. 4. Reduction of DPN by the supernatant fraction in presence of fructose-1,6-diphosphate and ADP was 0.1 to 0.2 micromoles per minute per gm. wet eggs, indicating that the glycolytic pathway can metabolize glucose-6-phosphate at about 5 per cent the rate at which it can be oxidized by the TPN system from unfertilized or fertilized Arbacia eggs. 5. Phosphoglucomutase, hexose isomerase, and a phosphatase for fructose-1,6-diphosphate also appear to be present in Arbacia eggs.     

THE EFFECTS OF URETHANE AND CHLORAL HYDRATE ON OXYGEN CONSUMPTION AND CELL DIVISION IN THE EGG OF THE SEA URCHIN,ARBACIA PUNCTULATA

The effects of a series of concentrations of the narcotics, ethyl carbamate and chloral hydrate, have been determined on the consumption of oxygen by fertilized and unfertilized eggs of the sea urchin Arbacia punctulata. In the fertilized eggs the effects of the two inhibitors on cell division were also examined. The following observations were made: 1. Assuming that the narcotic acts upon a single catalyst in the unfertilized egg the degree to which the consumption of oxygen is inhibited in this resting cell can be related to the narcotic concentration by an expression derived from the law of mass action. 2. To account for the relation between the concentration of the narcotic and its effect on respiration in the fertilized eggs, it is necessary to conclude that in them the narcotic acts on two parallel respiratory systems. The experimental data can be quantitatively predicted (1) if the reaction of the narcotic on the two systems is governed by the law of mass action and (2) if 40 per cent of the oxygen consumption is mediated by one system, the "activity" system, and the remainder by the other, the "resting" or "basal" system. 3. The mass law constants applying to the resting system in the fertilized egg are similar to those for the single system functioning in the unfertilized egg so that these two respiratory systems are probably identical. 4. The concentrations of the narcotics just sufficient to abolish cell division affect primarily the activity system, the existence of which was inferred from the respiratory experiments. It is concluded that normal cell division requires specifically the normal function of the activity system, that in fact the energy for cell division is made available through that system.     

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.     

Dinitrocresol and phosphate stimulation of the oxygen consumption of a cell-free oxidative system obtained from sea urchin eggs

1. A cell-free system capable of using oxygen with oxalacetate as substrate has been prepared from both unfertilized and fertilized sea urchin eggs. The oxygen uptake by this system is about twice that of an equivalent quantity of intact unfertilized eggs and half that of an equivalent quantity of intact fertilized eggs. 2. The oxygen consumption of this cell-free oxidative system can be stimulated by addition of suitable concentrations of 4,6-dinitro-o-cresol or by inorganic phosphate. This confirms, with a cell-free system obtained from sea urchin eggs, the observations of Loomis and Lipmann regarding stimulation of oxygen consumption by a system obtained from rabbit kidney. 3. A preliminary but unsuccessful attempt has been made to determine the conditions under which cell-free, aerobic, phosphorylating systems may be obtained from either unfertilized or fertilized sea urchin eggs.     

On the synthesis of glutamine before and after fertilization of the sea urchin, Strongylocentrotus purpuratus

Unfertilized eggs of the sea urchin, Strongylocentrotus purpuratus, have a much lower capacity for glutamine synthesis than do fertilized eggs. This difference is not caused by an alteration of glutamine synthetase activity attendant upon fertilization. Neither the specific activity of glutamine synthetase nor its pattern of activation by divalent metal ions is affected by fertilization. The enzyme from both fertilized and unfertilized eggs is activated by α-ketoglutarate and inhibited by ultimate end products of glutamine metabolism. This type of regulation is similar to that seen with many other eucaryotic glutamine synthetases.Unfertilized eggs take up less glutamic acid than do fertilized eggs when the amino acid is presented at high concentrations (12.5 mM), whereas there is no difference in glutamic acid uptake at low concentrations (5 μM). Under conditions where glutamate uptake is identical, unfertilized eggs are dependent upon exogenous ammonia for glutamine synthesis in vivo; fertilized eggs are able to synthesize glutamine in the absence of added ammonia. Thus, our data suggest that the increased capacity for glutamine synthesis after fertilization is related to an increased availability of the substrate, ammonia.     

Electrophoretic analysis of the stored histone pool in unfertilized sea urchin eggs: quantification and identification by antibody binding

A maternal store of histones in unfertilized sea urchin eggs is demonstrated by two independent criteria. Stored histones are identified by their ability to assemble into chromatin of male pronuclei of fertilized sea urchin eggs in the absence of protein synthesis, suggesting a minimum of at least 25 haploid equivalents for each histone present and functional in the unfertilized egg. In addition, electrophoretic analysis of proteins from acid extracts of unfertilized whole eggs and enucleated merogons reveals protein spots comigrating with cleavage stage histone standards, though not with other histone variants found in later sea urchin development or in sperm. Quantification of the amount of protein per histone spot yields an estimate of several hundred haploid DNA equivalents per egg of stored histone. The identity of some of the putative histones was verified by a highly sensitive immunological technique, involving electrophoretic transfer of proteins from the two-dimensional polyacrylamide gels to nitrocellulose filters. Proteins in amounts less than 2 x 10(-4) micrograms can be detected by this method.     

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 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.     

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.     

Insulin receptor sites as membrane markers during embryonic development. I. Data obtained with unfertilized and fertilized sea urchin eggs.

Binding of insulin to sea urchin egg plasma membrane has been studied by biochemical and immunocytochemical methods. Unfertilized and fertilized eggs as well as embryos during the first cell division have been used. 1. Competition experiments between 125I-insulin (1 nM) and an excess of native insulin (30 muM) indicate a specific hormone fixation to membrane crude extracts from unfertilized and fertilized eggs. The magnitude of "specific binding" is comparable to values recorded for mammalian cells. 2. Inhibition of insulin fixation by concanavalin A (100 mug/ml) suggests the glycoprotein composition of plasma membrane receptors. 3. An 30-min incubation of unfertilized and fertilized eggs in the presence of insulin leads to a significant increase in cyclic AMP content. 4. An immunocytochemical method demonstrates that insulin is selectively and specifically bound to the plasma membrane of eggs incubated in the presence of insulin before fixation. It can be concluded that insulin receptor sites are components of sea urchin eggs plasma membrane. Insulin binding which leads to cyclic AMP accumulation is not deeply modified by fertilization and does not include visible morphological changes in the eggs.     

Metal-binding proteins in eggs of various sea urchin species.

Metallothionein presence and amount were determined in the unfertilized eggs of six sea urchin species by silver saturation assay and gel-chromatography of cell extracts. The results showed high levels of metallothionein in the egg cytoplasm of the two Mediterranean species Paracentrotus lividus and Sphaerechinus granularis. No metallothionein was found either in the eggs of Arbacia lixula, or in those of the three Eastern species Strongylocentrotus intermedius, Temnopleurus hardwickii and Clypeaster japonicus. However, the extracts of the latter three species revealed the presence of zinc bound in a macromolecular form, thus suggesting the existence of metal-binding proteins distinct from metallothioneins.     

Potentiation of vinblastine crystal formation in vivo by puromycin and colcemid

The quantity of microtubular protein present in unfertilized eggs and zygotes of the sea urchin, Lytechinus pictus was assayed using vincaleucoblastine (VLB). This vinca alkaloid, used at 10^-4 M, induces the formation of highly birefringent crystals (uniaxial, refractive index 1.51, coefficient of BR 1.7 × 10^?3) known to contain microtubular protein. Crystals were measured in vivo with an ocular micrometer and the volume of each crystal was estimated. Total crystal volumes per cell were compared among groups which received VLB alone and other groups which received VLB with puromycin, colcemid or both. Puromycin was shown to have no effect on the VLB crystal volume in the unfertilized eggs, but zygotes incubated with VLB plus puromycin had a larger volume of crystals per cell than zygotes incubated in VLB alone. Colcemid (10^?4 M) clearly and consistently enhanced the volume yield of VLB crystals by a factor of 3 in both unfertilized eggs and zygotes.     

Localization of tropomyosin in sea urchin eggs

Localization of tropomyosin in sea urchin eggs was investigated immunohistochemically. A rabbit antiserum against tropomyosin prepared from lantern muscle of the sea urchin was used for the indirect immunofluorescence staining of unfertilized and fertilized eggs. The tropomyosin-specific fluorescence was observed at the peripheral region beneath the plasma membrane, mitotic apparatus and contractile ring. The mitotic apparatus isolated from sea urchin eggs was also stained with the anti-tropomyosin serum.     

Fertilization alters the orientation of pigment granule saltations in Arbacia eggs.

Unfertilized eggs of the sea urchin Arbacia punctulata contain pigment granules distributed throughout their cytoplasm. During the first 15 minutes after fertilization, these vesicles move out to the cortex where they become firmly anchored. We have used time-lapse video differential interference microscopy to analyze the motility of these organelles in unfertilized and fertilized Arbacia eggs. Pigment granules exhibit saltatory movement in both unfertilized and fertilized eggs. Quantitation of vesicle saltations before and after fertilization demonstrates that while there is no significant difference in the speed or path-length of vesicle movement, there is a dramatic change in the orientation of these saltations. Saltations in the unfertilized egg are very non-radial and are as likely to be directed toward the cortex as away. In contrast, saltations in the fertilized egg are more radially oriented and more likely to be cortically directed. This transition must reflect underlying changes in the cellular structures necessary for pigment granule saltations. The change in the orientation of pigment granule saltations following fertilization requires both a transient increase in the cytoplasmic concentration of Ca2+ and an elevation of cytoplasmic pH. Similarly, the ability of pigment granules to adhere to the cortex requires both the transient elevation of cytoplasmic Ca2+ and the alkalinization of the cytoplasm. As the reorganization of cortical actin at fertilization is regulated by these ionic fluxes, and both movement and adhesion are sensitive to cytochalasins, we hypothesize that the alterations in directed motility and adhesion reflect underlying changes in the actin cytoskeleton.     

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.     

Respiration capacity of mitochondria isolated from unfertilized and fertilized sea urchin eggs

Mitochondria were isolated from unfertilized and fertilized eggs of the sea urchin, Strongylocentrotus purpuratus. Both preparations exhibited coupled adenosine 5'-diphosphate (ADP)-dependent) oxidation of flavin and pyridine-linked substrates and both yielded the expected P:O ratios with these substrates. Highest respiratory control indices (greater than 4.0) were observed when succinate or pyruvate + malate were used as substrates. Mitochondria from unfertilized and fertilized eggs exhibited sensitivity to respiratory and phosphorylation inhibitors and uncouplers and both preparations exhibited cross-over points at sites I, II and III of the respiratory chain. Low-temperature difference spectra revealed a normal complement of cytochromes c, b and aa3, although cytochrome c from unfertilized eggs appears to be more subject to extraction during the course of mitochondrial isolation than does cytochrome c from fertilized eggs. An unidentified pigment absorbing at approx. 570 nm was visible in low-temperature spectra of unfertilized eggs and unfertilized egg mitochondria.     

THE MEMBRANE CAPACITANCE OF THE SEA URCHIN EGG

1. The surface of the unfertilized sea urchin egg is folded and the folds are reversibly eliminated by exposing the egg to hypotonic sea water. If the plasma membrane is outside the layer of cortical granules, unfolding may explain why the membrane capacitance per unit area decreases (and does not increase) when a sea urchin egg is put into hypotonic sea water. 2. The degree of surface folding markedly increases after fertilization, which provides an explanation for the increase in membrane capacitance per unit area observed after fertilization. 3. The percentage reduction in membrane folding in fertilized eggs after immersion in hypotonic sea water is probably sufficient to explain the decrease in membrane capacitance per unit area observed in these conditions.     

ON THE RATE OF OXYGEN CONSUMPTION BY FERTILIZED AND UNFERTILIZED EGGS : IV. CHAETOPTERHS AND ARBACIA PUNCTULATA

1. Unfertilized eggs of Chaetopterus consume about 2.4 mm.³ O₂ per hour per 10 mm.³ eggs at 21°C. 2. In the 1st hour after fertilization, the fertilized eggs consume oxygen at about 53 or 54 per cent of this rate, which is about 1.3 mm.³ O₂ per hour per 10 mm.³ eggs at 21°C. 3. For the first 6 hours after fertilization, at 21°C., the curve of the rate of oxygen consumption is slightly asymmetrically sigmoid. The prefertilization rate is regained between 4½ and 5 hours after fertilization. Soon after 6 hours, ciliary activity begins, and the rate of oxygen consumption rises rapidly. 4. The unfertilized eggs of Arbacia punctulata consume about 0.36–0.5 mm.³ O₂ per hour per 10 mm.³ eggs at 21°C. The absolute determination is difficult as these eggs are highly sensitive to shaking in the manometer vessels, and these difficulties are discussed. 5. The fertilized eggs of Arbacia punctulata consume oxygen at the rate of about 2.0 mm.³ O₂ per hour per 10 mm.³ 21°C. At 1 hour after fertilization the rate is already rising. 6. A comparison of the absolute rates of oxygen consumption, and the changes in rate at fertilization of these and a number of other eggs, together with a theoretical discussion, and a discussion of discrepancies in measurements on the eggs of Arbacia punctulata, is contained in the fifth paper of this series (21).     

Respiration of the tissues of some invertebrates and its inhibition by cyanide

A study of the metabolism of Bermuda marine invertebrates at 25 degrees C. shows that the respiratory rates of many of the tissues approximate those of vertebrate tissues at the same temperature. There is no apparent correlation between respiratory rate and phylogenetic development: tissues from some of the simpler forms use as much oxygen per unit weight as those from certain of the more highly developed animals. Cyanide inhibition experiments reveal a great variation in the amount of oxygen consumption which is dependent upon sensitive heavy metal systems. Three types of tissues, the jellyfish Cassiopea frondosa, the branchial tree of the sea cucumber, Stichopus m?bii, and two kinds of tunicates, were completely unaffected by even 10(-2)M HCN. Other tissues such as sea urchin sperm, squid gills, and lobster nerve and muscle were almost completely inhibited by much lower concentrations. Most of the materials retained 20 to 40 per cent of the normal respiratory rate in 10(-2)M HCN. The possibility that vanadium may play a part in the oxidation-reduction systems of the completely resistant animals is discussed. There is a thousandfold variation in the concentration of cyanide required to produce 50 per cent inhibition of respiration in the different tissues. Sea urchin sperm is 50 per cent inhibited by 10(-6)M HCN: the sea fan requires 10(-3)M for the same effect. Other tissues lie at intermediate points. When the logarithm of the ratio of the inhibited to the uninhibited respiration is plotted against the concentration of cyanide the resulting line has a slope which in most cases approximates 1. This indicates that one mole of enzyme ordinarily combines with one mole of inhibitor. Eggs of the sea urchin, Tripneustes esculentus, show a three- to fivefold increase in the rate of oxygen uptake on fertilization. The respiration of both the fertilized and unfertilized eggs is almost entirely inhibited by 10(-4)M HCN. Cell division in the fertilized eggs is blocked by somewhat less than 10(-5)M cyanide, a concentration which reduces respiration to 40 per cent of the normal level.