Egg Homogenates Research Articles

Change in the calcium-binding capacity of sea urchin egg homogenate caused by treatment with ATP and magnesium ions

The dissociation constant of the calcium and calcium-binding substance complex in sea urchin egg homogenate increased during incubation of the homogenate with CaCl 2 at concentrations greater than 0.5 mM to the same value as that found in the fertilized egg homogenate. The dissociation constant of the calcium-binding substance in the unfertilized egg homogenate (pre-incubated with CaCl 2), as well as the homogenate of the fertilized egg, was found to decrease to the value found in the unfertilized egg homogenate on treatment with 5 mM ATP and 5 mM MgCl 2. It was found that a calcium-binding substance of the ‘unfertilized egg type’ was easily transformed to a ‘fertilized egg type’ by calcium ions and the fertilized egg type substance reversibly became an unfertilized egg type on treatment with ATP and Mg ions. This substance is therefore thought to play an important role in the regulation of the intracellular concentration of calcium.

Endosymbionts: Factors of egg pattern formation

Elimination of the intracellular symbionts of Euscelis plebejus either by X-ray irradiation of the posterior pole of the freshly laid egg or by interruption of egg infection by application of tetracycline or lysozyme to female leafhoppers leads to the production of embryos without abdomens, ‘head-embryos’. Homogenates of symbiont-free eggs and symbiont-containing eggs in the state of invagination have a pH of 7·5±0·2 and 7·0±0·2 and an osmotic pressure (pO) of 8·3±0·2 and 7·8±0·2, respectively. The presence of symbionts leads to a decrease of both the pH and pO. These data indicate that the correct formation of the posterior gradient, necessary for normal abdomen development, is dependent on the presence of endosymbionts at the posterior pole. It is possible that the symbionts change the pH and pO of the posterior gradient. These results are consistent with a hypothetical model of early differentiation of the Euscelis egg.

The control of egg maturation by juvenile hormone in the tobacco hornworm moth, Manduca sexta.

1. In Manduca sexta females, follicles develop independently of the corpora allata until they reach a critical stage in late vitellogenesis. In the absence of juvenile hormone (JH), follicles then degenerate. When JH is present, the follicles continue development; they then become independent of JH for the final 12 hours of egg maturation. 2. Allatectomy of the pupa of Manduca sexta or the newly eclosed adult female prevents egg maturation as does extirpation of the brain from the pupa or decapitation of the adult. The ability to develop follicles beyond the vitellogenic phase is restored by injection of synthetic JH or JH mimics. 3. The brain activates the corpora allata close to the time of adult emergence. The glands secrete JH at a constant rate for 48 hours and then become less active in virgin, unfed moths. 4. A female specific protein, identified by SDS gel electrophoresis, is present in adult blood and egg homogenates. Allatectomy has no effect on the concentration of vitellogenin in the blood nor on its incorporation into immature follicles less than one mm long. 5. The action of JH in promoting egg maturation in Manduca sexta appears to be different from that in most other well-studied insect systems in that the hormone is not required for the synthesis and/or uptake of the female-specific yolk protein. Rather it is required for a critical step in development.

Activation of sea-urchin eggs by a calcium ionophore.

Micromolar amounts of the divalent ionophore A23187 can activate echinoderm eggs. The activations by ionophore A23187 were examined in terms of membrane elevation, the program of membrane conductance changes, the respiratory burst, and the increases in protein and DNA synthesis which normally accompany activation by sperm. In all these respects activation by the ionophore was fairly normal although subsequent cleavage and embryonic development was limited. Ionophore A23187 activations of the cortex of Lytechinus pictus and Strongylocentrotus purpuratus eggs were compared in various ionic media and were found to be completely independent of the ionic composition of the external solution. Respiration and protein synthesis of L. pictus eggs in singly substituted ionic media also indicated that these activations were independent of external sodium, calcium, or magnesium. These results suggest that the ionophore acts by releasing intracellular Ca(++). Consistent with this interpretation is the finding that eggs preloaded with (45)Ca show a 20-fold increase in (45)Ca-efflux when activated by ionophore A23187 or fertilization. Measurements of the "free" and "bound" calcium and magnesium in homogenates of the unfertilized eggs show that most of the Mg(++) is already available in the soluble form, whereas Ca(++) is sequestered but available for release. We propose that both normal fertilization and ionophore activation affect the metabolism of the egg by releasing Ca(++) sequestered in intracellular stores.

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 CaCl(2) 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.

Functional volume of frog eggs: Equivalence of metabolite diffusion space in chemically demembranated embryos and aqueous phase (non‐yolk) volume

AbstractVitelline membranes were removed from two‐cell Rana pipiens embryos by papain‐cysteine. Uptake of several different metabolites was enhanced, some more than others, but they all slowly reached about the same plateau, 11.2–13% (average) volumetric equilibration of the embryos with the radioactive medium. Active transport was ruled out as the uptake mechanism.Similarly, direct measurement of the volume of the aqueous phase, or “non‐yolk” cytoplasm, of egg homogenates gave an average value of 12.3% of the total egg volume. We conclude that this represents the available embryonic diffusion space and that the metabolites saturated it by passive diffusion. The importance of the concept of non‐yolk cytoplasm, or egg functional volume, as a developmentally significant parameter is discussed.

Maternal mRNA and protein synthesis in the embryo.

ABSTRACT According to the masked maternal messenger RNA hypothesis, a large part if not all the protein synthesis of early development is directed by mRNA already present in the cytoplasm of unfertilized eggs. This mRNA is supposed to be synthesized during oogenesis and stored in some unavailable form until some later time in development, when it is selectively associated with the translational machinery. To the indirect evidence, which is nevertheless very strong, there can now be added a direct proof of the hypothesis for the case of histone mRNA. The five main histones of sea urchin embryos are synthesized on small polyribosomes, directed in part by newly-synthesized messages that sediment as a group at about 9S. Some histone synthesis survives total transcription block, however, suggesting that maternal histone mRNA exists. In competition-hybridization experiments, the egg RNA is shown to contain sequences characteristic of functional, embryonic histone mRNA. The competing RNA is localized in ribonucleoprotein particles of egg homogenates that sediment at 20–40S. These same particles contain RNA that stimulates a cell-free heterologous system to synthesize sea urchin histones. The application of these facts to some problems of translation control and of development generally is discussed.

Activation of phosphorylase in sea urchin eggs by Ca 2+ 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 Ca 2+ showed the maximal activating effect on the AMP-independent form of phosphorylase. The fertilization-induced increase of Ca 2+ and of cAMP were discussed as possible activators of phosphorylase, and consequently, of carbohydrate metabolism.

Regulation of Tubulin Organization During Meiosis

Meiotic spindle formation in Spisula solidissima oocytes has been studied in vivo and in vitro. Measurements were made of polymerized tubulin content during the first meiotic division. The amount of polymer was high prior to activation of the eggs, fell to a minimum of about 5 min after activation and at 15 min (metaphase) returned to approximately its initial value. The polymerized tubulin in the unactivated eggs appears to be organized into granular spheres about 10–20 microns in diameter attached to the egg cortex. This particle contains few microtubules but is composed primarily of a granular matrix and fibrous material. The granular matrix may be a polymorphic aggregate of tubulin and could be a storage form of tubulin or an intermediate in microtubule assembly. The polymerization and organization of microtubules has been studied in vitro, using crude homogenates of Spisula oocytes. Microtubules can be formed in homogenates of both activated and unactivated eggs; however, in homogenates of eggs in which nuclear membrane breakdown has occurred, microtubules form around a central phase dense particle resulting in a structure which resembles a spindle aster. The central particle appears to be a microtubule organizing center (MTOC). The MTOC can be pelleled by centrifugation and will induce aster formation when remixed with the supernatant. Aster formation can be obtained using supernatants prepared from either activated or unactivated eggs, while the pellet must be obtained from activated eggs. Thus, tubulin subunits appear to be capable of spindle formation at all stages, while MTOC formation or activation does not occur until about the time of nuclear membrane breakdown.

A comparative study of selected tissues from the parasitod Cardiochiles nigriceps, its susceptible host Heliothis virescens and a resistant host Heliothis zea by polyacrylamide gel electrophoresis

1. 1. Fourteen anodically migrating protein bands were observed in the hemolymph of both Heliothis zea and H. virescens using polyacrylamide gel electrophoresis while the calyx fluid from Cardiochiles nigriceps showed very dilute protein concentrations. 2. 2. In vitro and in vivo melanized blood samples from H. zea and H. virescens contained eleven observable bands. 3. 3. Superparasitized H. zea showed a reduction in hemolymph proteins with eight observable bands. 4. 4. Electrophoresed materials from the parasite, C. nigriceps, revealed eleven protein bands from the teratocytes, nine protein bands from egg homogenates, nine protein bands from second instar larvae homogenates and seven protein bands from the calyx fluid.

A trypsin-like proteinase localized in cortical granules isolated from unfertilized sea urchin eggs by zonal centrifugation. Role of the enzyme in fertilization

A trypsin-like proteinase was localized within a single subcellular compartment of unfertilized Strongylocentrotus purpuratus eggs, the cortical granules. Homogenates of eggs were fractionated by rate-zonal centrifugation. Enzymatic markers were used to determine the distribution of mitochondria (cytochrome oxidase), yolk platelets (acid nitrophenyl phosphatase), and cortical granules (β-1, 3-glucanase) in the sucrose density gradient. A bimodal distribution pattern was obtained for aryl esterase activity (substrate: β-naphthyl acetate), with one peak in the microsomal and the other in the cortical granule fractions. The cortical granule enzyme was characterized as a trypsin-like proteinase, since it also hydrolyzed another typical tryptic substrate α- N-benzoyl- l-arginine ethyl ester and was completely inactivated by soybean trypsin inhibitor (SBTI). The aryl esterase activity in the microsomal fractions was not inhibited by SBTI, while 50% of the total aryl esterase activity in the original egg homogenate was inactivated by SBTI. The identity of the enzyme(s) responsible for the aryl esterase activity associated with the microsomal particles is unknown at present. The cortical granule proteinase functions in the elevation of the fertilization membrane and establishment of the block to polyspermy at fertilization. Arbacia punctulata eggs inseminated in the presence of trypsin inhibitors, SBTI or tosyl lysine chloromethyl ketone (TLCK), failed to elevate normal fertilization membranes and became heavily polyspermic. On the basis of these results and observations made by other investigators with a wide variety of biological systems, it is proposed that trypsin-like proteinases function in the discharge of secretory granules from all types of cells.

Studies on metabolism of lung fluke genus Paragonimus. VII. Action of bithionol on glycolytic and oxidative metabolism of adult worms

The present study was carried out in order to obtain information on the mechanism of action of bithionol on Paragonimus westermani (Kerbert 1878). Bithionol stimulated lactic acid production of intact adult worms above the level of the control worms, while it inhibited oxygen consumption of intact adult worms in vitro. Bithionol treatment of adult worms in vivo decreased glycolytic and oxidative metabolism of homogenates of uterine eggs and adult worms. Bithionol inhibited lactic acid formation except when fructose 1, 6-diphosphate (FDP) was used as a substrate in the homogenates of adult worms, and it also inhibited oxygen consumption of homogenates of eggs and adult worms. Bithionol inhibited reduction of methylene blue when succinate was used as a substrate. Bithionol inhibited oxidation of reduced cytochrome c in the 1000 g supernatant of homogenates of adult worms. Bithionol inhibited activity of the succinate oxidation in homogenates of adult worms.

Studies on metabolism of lung flukes of the genus Paragonimus VI. Succinoxidase system in homogenates of eggs, larvae, and adults

The present study was concerned with the succinoxidase system in Paragonimus westermani, Paragonimus ohirai, and Paragonimus miyazakii. Potassium cyanide inhibited the motility of larval and adult forms. Succinate stimulated the reduction of methylene blue by homogenates of embryonated eggs, larvae, and adults, while malonate inhibited the reduction. Reduced cytochrome c was oxidized by the 1,000 g supernatant from homogenates of embryonated eggs, larvae, and adults. The supernatant prepared from unembryonated eggs did not oxidize reduced cytochrome c. Succinate stimulated oxygen consumption by the homogenate of adult worms. Oxygen consumption markedly increased in the homogenate of adults when both succinate and cytochrome c were added as substrate to the reaction mixture, while malonate and cyanide inhibited oxygen consumption.

Redistribution of tosylarginine methylester hydrolase activity after fertilization of sea urchin (Arbacia punctulata) eggs.

Low speed residues (750g) from unfertilized sea urchin egg homogenates contain an enzyme capable of hydrolysing tosylarginine methylester (TAME)1. Enrichment of this particulate fraction by differential centrifugation, followed by discontinuous sucrose gradient centrifugation, made it possible to distinguish visually about seventeen bands within the gradient (Fig. 1). Each band was carefully removed with a Pasteur pipette and assayed for total TAME hydrolase activity. As shown in Fig. 1, most of the enzyme was in a single narrow zone (fraction 5). This fraction banded at the interface between 1.00 and 1.55 M sucrose, indicating that the particles contained within this region of the gradient had a density of 1.14–1.19. When a similar fractionating procedure was carried out with 5-min-old sea urchin embryos TAME hydrolase activity in the particulate fraction isolated by differential centrifugation decreased to about 30% of that found in unfertilized egg preparations. As the particulate fraction contains 80% or more of the total TAME hydrolase activity in unfertilized eggs, there must be considerable redistribution of this enzyme to account for the 70% loss in activity of this fraction after fertilization.

An enzymatic comparison of sea urchin egg ghosts prepared before and after fertilization

AbstractGhosts were prepared from sea urchin eggs, before and after fertilization. Eggs were treated with Mg++ to stabilize the surface. Cytoplasm was then released by osmotically lysing the cells and washing repeatedly at low ionic strength. Microscopically, the resultant ghosts appear to contain the extraneous surface coats, some cell cortex, and the plasma membrane, but relatively little cytoplasm. This appearance and the higher specific activities of Na+‐K+‐ATPase, Mg++‐ATPase, and acetylcholinesterase in ghosts than in whole egg homogenates, indicates the usefulness of ghost preparations as egg surfaces in enzyme comparison studies. The specific activities of five of the six enzymes measured are different when compared in ghosts prepared before and after fertilization. Glucose‐6‐phosphate‐dehydrogenase, acetylcholinesterase, and Na+‐K+‐ATPase activities are greater in fertilized than in unfertilized egg ghosts, whereas the Mg++‐ATPase and glucose‐6‐phosphatase activities are lower. The changes in enzyme specific activities following fertilization may be related to structural and functional changes which occur in the egg surface at that time.

Inhibition of ciliary movement by sea urchin egg homogenate

1. 1. Homogenates of unfertilized eggs of sea urchins and some subcellular fractions from them inhibit the ciliary movement of sea urchin embryos. 2. 2. Inhibition by the homogenate of sea urchin eggs is specific to the ciliary movement of various sea urchin and sand dollar embryos but not to the ciliary movement in other animals tested. 3. 3. The inhibitory factor in the homogenate is thermostable, nondialyzable, acid-extractable, alkali-precipitable and trypsin digestible. 4. 4. These findings together with the other observations seem to suggest that the egg factor inhibiting the ciliary movement in sea urchin embryos may be related to a basic protein or a polypeptide.

Changes in activity of proteases during embryogenesis of Anagasta kuehniella (insecta)

1. 1. Cleavage of hemoglobin by homogenates of eggs of Anagasta kuehniella (Zeller) has been investigated. Three distinct pH optima (4·5, 6·9 and 9·5) of activity are manifested during embryological development. Several characteristics suggest the two activities with lower pH optima are of the cathepsin type while activity at the high pH may be of the trypsin type. 2. 2. The activity having optimum at pH 6·9 fluctuates in a diurnal manner during the 4-day development period. Such diurnal fluctuations in activity persisted even in eggs in which embryonic development had been arrested by exposure to 57°C for 15 min within 6 hr after oviposition. 3. 3. The protein complement undergoes virtually complete alteration during embryogenesis. Loss of soluble proteins from the egg near the end of the developmental period seems to correlate with increases in proteolytic activity.

Regulation of onset of rapid hemoglobin formation in chick blastodisc blood islands at the transcription and translation levels. A quantitative analysis of effects of actinomycin, ethanol, and heme precursors.

Amounts of hemoglobin formed by blastodiscs explanted for 24 h on minimal medium containing 2 μg/ml of actinomycin were independent of initial stage of development between the 2- and 6-somite stages. No additional hemoglobin was formed by 2- to 4-somite blastodiscs in response to further supplements of aminolevulinate. In contrast, levels of hemoglobin formed in the presence of actinomycin by both younger and older blastodiscs were dependent on initial stage of development, and also were enhanced by aminolevulinate. Protoporphyrin both stimulated formation of hemoglobin on minimal medium to levels comparable with those formed on egg homogenate medium, and prevented inhibition by actinomycin of the synthesis of macromolecules. Ethanol in concentrations up to 1% had no effect on levels of hemoglobin formed or incorporation of precursors into macromolecules.On egg homogenate medium low concentrations of ethanol markedly stimulated hemoglobin formation, probably in part through induced synthesis of aminolevulinate synthetase enzyme. High concentrations of ethanol also inhibited incorporation of leucine into polypeptide. Incorporation of uridine into polynucleotide was only partially inhibited on egg homogenate medium containing 2 μg/ml of actinomycin. Hemoglobin formation was markedly less inhibited than on minimal medium and was stimulated by both aminolevulinate and protoporphyrin.

Purification of cortical granules from unfertilized sea urchin egg homogenates by zonal centrifugation

Cortical granules were purified from homogenates of unfertilized sea urchin, Strongylocentrotus purpurataus, eggs by rate-zonal centriguation. Purification was evaluated by integrated biochemical and morphological (electron microscopic) analysis. The enzyme β-1,3-glucanase was employed as the biochemical marker for cortical granules. Cortical granule preparations obtained in sucrose gradients constructed in 0.5 M KCl contained acid phosphatase activity and were heavily contaminated by yolk platelets. Highly purified cortical granules isolated in sucrose gradients constructed in 0.5 M KCl with 10 −3 M EDTA at pH 7.0 were almost completely free of contamination by yolk and other cytoplasmic particles, and contained only trace levels of acid phosphatase activity. The purified cortical granules contained 40–45% of the total β-1,3-glucanase activity in the eggs, and 6.7 ± 1.9% of the total egg protein. The isolated cortical granules retained another known chemical constituent, sulfated acid mucopolysaccharides.

Selective inhibition of hemoglobin formation in chick embryo blood islands by chloramphenicol.

Chloramphenicol markedly inhibited the formation of hemoglobin by chick blastodiscs explanted before the 8-somite stage of development onto minimal or egg homogenate medium. Sensitivity to inhibition of hemoglobin formation was reduced at the 8-somite stage. Chloramphenicol had no effect upon the incorporation of leucine into total protein, and only slightly inhibited the cycloheximide-resistant incorporation of leucine.