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Fertilization of immature frog eggs: cleavage and development following subsequent activation.

Frog eggs are normally fertilized after reaching metaphase II. When eggs are inseminated prior to that, several sperm enter, but entry does not activate the egg. When such inseminated, immature eggs were maintained until they became mature and then were artificially activated, the eggs began to cleave. The cleavage furrows were irregular and often multiple, but the eggs developed to blastulae or partial blastulae. About 2 leads to 5% of the eggs developed to tadpoles. Typical asters were not associated with the entering sperm; rather, asters appeared only after activation. The sperm nucleus often formed chromosomes which were attached to small spindles. It is clear that sperm which remain for a time in unactivated egg cytoplasm, retain their ability to promote cleavage and development. Aster formation required not only sperm centrioles but also activated egg cytoplasm. Sperm which entered either near the equator or in the animal half of mature eggs usually produced normal cleavage furrows. Sperm which entered the animal half of immature eggs produced multiple animal half furrows when the egg was subsequently activated. In contrast, sperm which entered near the equator of immature eggs often failed to induce furrowing on subsequent activation or produced unusual equatorial furrows. The difference in the type of furrow between eggs inseminated in the animal half or at the equator is interpreted as a consequence of dissociating sperm entry from the cortical contraction which occurs in activation.

Animals

Glutathione reductase in the sea urchin egg. III. Activation of the complex form by proteinases.

The G-200 flow-through fraction of the extract of sea urchin eggs contained a complex form of glutathione reductase (GR) [EC 1.6.4.2]. The complex was unstable and gradually dissociated with ain increase in GR activity. The activation was facilitated by high concentrations of EDTA, KCI or (NH4)2SO4. The rate of activation by salts was apparently dependent on the ionic strength. The complex form was also activated rather quickly by treatment with proteinases such as trypsin [EC 3.4.21.4], alpha-chymotrypsin [EC 3.4.21.1] or subtilisin [EC 3.4.21.14]. Trypsin caused the complex to release the free form of GR. Even after trypsin treatment, little change was observed in the dependence of the GR activity on GSSG or NADPH concentration. The GR activity of the complex form was not inhibited at all by 0.2 mM N-ethylmaleimide (NEM) in the presence of GSSG, but was reduced to 3% in the presence of NADPH. When excess NEM was sequestered with GSH, the NEM-treated complex form was strikingly activated by trypsin, while no activation was detected with the free form of enzyme pretreated with NEM. These results suggest that the active site of GR in the complex form is largely masked by a polypeptide moiety of theinhbitiory component.

Animals

Free calcium increases explosively in activating medaka eggs.

We have used the calcium-specific light-emitting protein aequorin to follow changes in free calcium concentration during fertilization and cleavage of eggs from medaka, a fresh-water fish. Aequorin-injected medaka eggs show a very low resting glow before they are fertilized, indicating a low calcium concentration in the resting state. Upon activation by sperm, the calcium-mediated light emission increases to a level some 10,000 times the resting level with a 1 to 2 sec time constant for an e-fold increase, and then slowly retruns to the resting level. Upon activation by the ionophore A23187, the early rise in luminescence is much slower, but once a threshold has been reached the subsequent rise becomes as rapid as the normal sperm-induced response. We infer that the explosive rise in calcium involves calcium-stimulated calcium release, and that a sperm normally triggers this rise by somehow inducing a more modest and localized rise in calcium.

Aequorin

Intracellular pH and the sodium requirement at fertilisation.

Several lines of evidence suggest that ionic messengers are primary agents in the metabolic derepression which occurs at fertilisation. The derepression at fertilisation or parthenogenetic activation of the sea urchin egg occurs in two main phases. The first phase, which triggers the early events of fertilisation, is mediated by transitory increase of intracellular free calcium. The second, which triggers the late events of fertilisation, is mediated by a rise in the intracellular pH (refs 4-6). The transition from the early events of fertilisation of sea urchin eggs to the late events requires a minimal concentration of sodium in the external medium. External Na+ is required for the acid effux which follows fertilisation. Na+ requirement and the acid effux have been correlated in a hypothesis which proposes that internal protons are exchanged for external Na+ (refs 8, 9). By using pH-sensitive microelectrodes, we have examined the relationship between external Na+ and internal pH more closely. We demonstrate here that the increase of the intracellular pH following egg activation does require external Na+. However, the relative insensitivity of the alkalisation of the egg cytoplasm to large reductions of external Na+ is evidence against the Na-H exchange hypothesis.

Allosteric Regulation

The role of lipid-protein interactions in NADH-cytochrome c reductase (rotenone-insensitive) of rat liver mitochondria.

The phospholipid depletion of rat liver mitochondria, induced by acetoneextraction or by digestion with phospholipase A2 or phospholipase C, greatly inhibited the activity of NADH-cytochrome c reductase (rotenone-insensitive). A great decrease of the reductase activity also occurred in isolated outer mitochondrial membranes after incubation with phospholipase A2. The enzyme activity was almost completely restored by the addition of a mixture of mitochondrial phospholipids to either lipid-deficient mitochondria, or lipid-deficient outer membranes. The individual phospholipids present in the outer mitochondrial membrane induced little or no stimulation of the reductase activity. Egg phosphatidylcholine was the most active phospholipid, but dipalmitoyl phosphatidylcholine was almost ineffective. The lipid depletion of mitochondria resulted in the disappearance of the non-linear Arrhenius plot which characterized the native reductase activity. A non-linear plot almost identical to that of the native enzyme was shown by the enzyme reconstituted with mitochondrial phospholipids. Triton X-100, Tween 80 or sodium deoxycholate induced only a small activation of NADH-cytochrome c reductase (rotenone-insensitive) in lipid-deficient mitochondria. The addition of cholesterol to extracted mitochondrial phospholipids at a 1 : 1 molar ratio inhibited the reactivation of NADH-cytochrome c reductase (rotenone-insensitive) but not the binding of phospholipids to lipid-deficient mitochondria or lipid-deficient outer membranes. These results show that NADH-cytochrome c reductase (rotenone-insensitive) of the outer mitochondrial membrane requires phospholipids for its activity. A mixture of phospholipids accomplishes this requirement better than individual phospholipids or detergents. It also seems that the membrane fluidity may influence the reductase activity.

Animals

The effects of passive antibodies to egg albumin on active immunity in lambs to Brucella abortus and egg albumin.

The long-term effects of colostrum on active immunity to two unrelated antigens are described. Lambs were fed with pooled colostrum--to equalise passive immunity--with or without added antibodies to egg albumin (Ea). There were significant breed differences in the response both to Brucella abortus measured at one month of age, and to Ea, measured at three months of age, although there was no significant correlation between the responses to the two antigens, either within or between breeds. Surprisingly, whereas antibodies to Ea caused a four-fold reduction in antibody production to B abortus, they did not affect the overall mean response to Ea. But the timing of the response to Ea was significantly affected, suggesting that the low persisting concentrations of antibody had caused qualitative changes in the response.

Animals

Phosphatase active antigens in sea urchin eggs and embryos. II. A comparison between the activities in unfertilized eggs and plutei.

Phosphatase activities in sea urchin eggs and plutei were investigated by means of histochemical staining of immunoprecipitates. Two protein fractions were obtained by extraction in a hypotonic medium and by detergent treatment of the residual pellet. Three distinctly different phosphatase activities were discerned, nucleoside diphosphatase (EC 3.6.1.6.), acid phosphatase (EC 3.1.3.2.) and alkaline phosphatase (EC 3.1.3.1.). The nucleoside diphosphatase activity, which was confined to one antigen, was present in both water soluble and detergent extracts and at roughly the same concentration in eggs and plutei. By means of a monospecific antiserum the immunological identify of this antigen was established in all instances. The acid phosphatase activity, which was displayed by ten detergent extracted antigens in eggs, was only found in five detergent extracted antigens in plutei. This decrease in number of enzyme active antigens was also reflected by a general decrease in number of enzyme active antigens was also reflected by a general decrease in activity as assessed by quantitative determinations. Furthermore, by means of absorbed antisera it was established that two or three of the acid phosphatase active antigens were "egg specific". Another acid phosphatase active antigen, which was common to both developmental stages, was investigated by a monospecific antiserum. While this antigen was found in both soluble fractions, it was only enzymatically active when extracted with detergent. Alkaline phosphatase active antigens were only found in the detergent extract of plutei. However, immunoprecipitates with this activity appeared both with antiserum against unfertilized eggs and with antiserum against plutei. This suggests that the egg contained the antigens in an enzymatically inactive form.

Acid Phosphatase

Change in the fructose 1,6-bisphosphatase activity in sea urchin eggs following fertilization.

The activity of fructose 1,6-bisphosphatase [EC 3.1.3.11] in sea urchin eggs decreased following fertilization. During the first 30 min after fertilization, the activity was considerably lower than that in unfertilized eggs, but by 30 min the activity was similar to that in unfertilized eggs. The enzyme activity in fertilized eggs, estimated in the presence of EGTA, was similar to that in unfertilized eggs. The activity in unfertilized eggs was reduced by Ca2+ at concentrations between 1 X 10(-5) M and 5 X 10(-3) M. Immediately after fertilization, the enzyme was insensitive to concentrations of Ca2+ lower than 2 X 10(-4) M, but the Ca2+ sensitivity of the enzyme recovered 30 min after fertilization. In the presence of Ca2+ at concentrations higher than 2 X 10(-4) M, the enzyme activity in unfertilized eggs was similar to that in fertilized eggs. Mg2+ restored the Ca2+-induced inhibition of fructose 1,6-bisphosphatase. 3-Phosphoglycerate and citrate hardly affected the enzyme activity, and AMP at concentrations above 10 mM inhibited it.

Adenosine Monophosphate

Effect of dietary DL-ethionine and/or DL-methionine on egg laying and activities of some cytoplasmic NAD linked-dehydrogenases and NADPH-producing enzymes in liver of Japanese quail, Coturnix coturnix japonica.

The effect of dietary DL-ethionine and/or DL-methionine on egg laying, and activities of some NAD linked-dehydrogenases and NADPH-producing enzymes in liver of Japanese quail, Coturnix coturnix japonica was investigated. A 0.30% DL-ethionine plus 0.30% DL-methionine supplemented diet reversed partially the egg laying inhibited by the diet with 0.30% DL-ethionine alone. No inhibitory effect on egg laying was observed for the diet supplemented with 0.30% DL-methionine alone. In marked contrast to the decreased activity of L-glycerol 3-phosphate dehydrogenase and malate dehydrogenase, significantly increased activity of lactate dehydrogenase was obtained for quail fed the DL-ethionine, and the DL-ethionine plus the DL-methionine supplemented diet, respectively. No marked changes in activities of these three dehydrogenases were obtained for quail fed the diet supplemented with DL-methionine alone. Although decreased activity was observed for all of the four NADPH-producing enzymes in quail fed the diet supplemented with DL-ethionine alone, the DL-ethionine plus DL-methionine, the smallest decrease was obtained for NADP-isocitrate dehydrogenase. The diet supplemented with DL-methionine alone induced markedly the respective activity of malic enzyme and glucose 6-phosphate dehydrogenase. These results indicate a relatively important function of NADP-isocitrate dehydrogenase for NADPH-production even under DL-ethionine toxicity and suggest complicated relationships between egg production and activities of enzymes associated with carbohydrate and lipid metabolism in quail liver.

Alcohol Oxidoreductases

Egg surface glycoprotein receptor for sea urchin sperm bindin.

Bindin is an insoluble protein coating the sperm acrosome process and mediating the adhesion of sperm to sea urchin eggs. Milligrams of bindin have been isolated. Here we report the identification, isolation, and partial characterization of a high molecular weight, trypsin-sensitive glycoprotein fraction from the sea urchin egg surface having species-specific affinity for bindin. This glycoprotein may be the egg surface receptor for bindin. The bindin receptor was released from 125-I-labeled eggs by parthenogenetic activation of eggs with ionophore A23187 in the presence of soybean trypsin inhibitor. The receptor has an isoelectric point of 4.02 and a molecular weight in sea water greater than or equal to 5 X 10(6), suggesting that it is an aggregate. It contains 34% neutral sugars, which are galactose and mannose.

Adhesiveness

Evidence for mediation of a neuronal interaction by a behaviorally active peptide.

Egg laying hormone, a peptide neurohormone with an approximate molecular weight of 6000, was isolated from the region of the abdominal ganglion of Aplysia that contains the neuroendocrine bag cells and purified by gel filtration chromatography, isoelectric focusing, and dialysis. A 1-min local application of egg laying hormone to the identified neuron R15 produced prolonged (greater than 1 hr) augmentation of impulse activity in this neuron. The distinctive quality and prolonged duration of the response are apparently identical to the previously described response to electrically elicited bag cell activity. The results provide evidence that egg laying hormone is the mediator of this prolonged neuronal interaction.

Animals

Acidic thiol proteinase activity of Schistosoma mansoni egg extracts.

Extracts of the eggs of the human blood fluke, Schistosoma mansoni, exhibit proteolytic activity which requires the presence of added thiol reagents or cyanide. The pH optimum for hydrolysis of Azocoll and cartilage proteoglycan is 4.8--5.2 and the molecular weight of the major component is 25--26,000. The effects of inhibitors suggest this activity belongs to the acidic thiol proteinase class, with a similarity to Cathepsin B. These proteinases may be involved in nutrition of the egg or sporocyst, in penetration of eggs or miracidia through host tissues, or in the immunopathology of schistosomiasis.

Animals

[Experimental studies on the symmetry reaction of axolotl oocytes and eggs: factors affecting the early appearance of the grey crescent following heat shock].

A heat-shock (36.8 degrees C for 10 min.) can induce the immediate appearance of the grey crescent in a high proportion of freshly laid fertilised or artificially activated Axolotl eggs, but only if that shock occurs within 1 h 30 min. following deposit of eggs (at 18 degrees C) and if eggs are not dejellied. On non-activated virgin eggs, the heat-shock is efficient during at least 3 hrs. This symmetry reaction can be induced on in vitro artificially matured oocytes, still protected by follicular cells. It is uncommon in oocytes taken from coelomic cavity or from the anterior part of the oviduct, but occurs with a high frequency as soon as the occytes are coated with the innermost and medium jelly layers.

Ambystoma

Proteinase activity in Ascaris suum eggs, hatching fluid, and excretions-secretions.

Hatching fluid and the excretions and secretions (E.S.) of hatched larvae of Ascaris suum revealed proteinase activity when assayed by 2 different procedures employing collagen or casein as substrates. Both assays apparently detected similar levels of proteinase activity in hatching fluid and E.S. of hatched larvae. The Anson (casein substrate) assay worked best in 0.05 M phosphate buffer, pH 8.0. The Azocoll (collagen substrate) assay worked best in 0.05 M borate buffer at pH 8.8. Azocoll assays done at temperatures ranging from 25 to 65 C revealed maximal proteinase activity at 55 C. Analysis of hatching fluid from 18-, 21-, and 28-day-old embryos and of extracts from sonicated 0- to 28-day-old developmental stages showed that proteinase activity increased markedly 18 days after embryonation had begun. Prior to the 18th day of embryonation proteinase levels were relatively low.

Animals

Phosphatase active antigens in sea urchin eggs and embryos. I. Substrate specificity, pH-optima and inhibitors.

Phosphatase activity in sea urchin embryonic antigens was investigated by histochemical staining of immunoprecipitates separated by two-dimensional (crossed) immunoelectrophoresis. Unfertilized eggs were homogenized in a hypotonic medium which solubilized cytoplasmic antigens. Antigens integrated in membranes or enclosed in particles were solubilized by detergent treatment of the residual pellet. Two different phosphatase activities were discerned in the unfertilized eggs, nucleoside diphosphatase (EC 3.6.1.6.) and acid phosphatase (EC 3.1.3.2.). Nucleoside diphosphatase activity was obtained in both the water soluble and detergent extracted protein fractions. This activity was confined to one antigen. Acid phosphatase acitivity on the other hand was almost exclusively obtained in the detergent extracted fraction and about ten distinct antigens displayed this activity. The nucleoside diphosphatase active antigen preferentially hydrolyzed purine nucleoside diphosphates and to a lesser degree triphosphates of these nucleosides. The acid phosphatase active antigens had a broader substrate specificity and hydrolyzed equally well beta-glycerophosphate and nucleotides. Both activities were essentially inactive at neutral or alkaline pH values. The activities were inhibited by p-choloromercuribenzoate and accordingly stimulated by cysteine. Tartrate and sodium fluoride, however, inhibited the acid phosphatase activity while nucleoside diphosphatase activity was either stimulated or not affected at all by these agents.

Acid Phosphatase