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Attack of sea urchin eggs by dogfish phagocytes: model of phagocyte-mediated cellular cytotoxicity.

To test whether lysosomal degranulation of phagocytes is associated with antibody-dependent cytotoxicity, eggs of Arbacia punctulata were used as targets for blood phagocytes of Mustelus canis. Eggs were coated with heat-aggregated dogfish IgM and exposed to phagocytes, and cytolysis of eggs was observed by Nomarski optics. Phagocytes adhered, degranulated, and raised fertilization membranes resembling those induced by sperm or ionophore A23187. Lysis was then observed as damage radiating from the point of phagocyte-egg contact. By 4 hr, coated eggs exposed to phagocytes released 8.9, 12.3, and 7.4% of total catalase (EC 1.11.1.6), beta-glucuronidase (EC 3.2.1.31), and superoxide dismutase (EC 1.15.1.1) into the medium. Cytotoxic enzyme release significantly exceeded that from uncoated eggs incubated with phagocytes or eggs alone (uncoated or coated). Because activated eggs release a neutral protease, it was considered possible that this enzyme might be responsible for autolysis of eggs. This possibility was excluded because (i) lysis of eggs was not inhibited by soybean trypsin inhibitor (SBTI) whereas the egg protease was sensitive to SBTI, and (ii) the major trypsin-like activity of phagocytes was not inhibited by SBTI. These experiments demonstrate that Ig-coated cells are first activated, and then killed, when exposed to degranulating phagocytes and suggest that enzymes from attacking phagocytes, and not target cells, are responsible for cell death.

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

Experimental activation of ascidian eggs.

The effects of the ionophore A23187 on the activation of the eggs of Ascidia malaca have been studied. No common external ion in the sea water is found to be essential for the activation but lanthanum and manganese inhibit the response. These observations support the interpretation that activation of these eggs results from changes in free intracellular calcium levels. This has led to the prediction of two other activating treatments, namely high external calcium and addition of theophylline.

Animals

Accelerated mouse sperm penetration in vitro in the presence of caffeine.

Evidence for accelerated mouse sperm penetration into eggs in vitro, and hence for accelerated capacitation, was obtained when the fertilizing ability of sperm suspensions was tested after preincubation, for a period of time insufficient to permit full capacitation under standard conditions, in the presence or absence of caffeine. When eggs were fixed after 1 h, the majority (76.7%) in the caffeine-containing medium were fertilized compared with only 32.1% in the caffeine-free medium. When fixation was later, after 1 h 15 min, fertilization levels were high (approximately 90%) in both groups, but significant differences in the stages of egg activation and sperm head decondensation reached were observed. In the absence of caffeine, early stages of nuclear development were predominant, while in the presence of caffeine, a significant proportion of eggs had reached terminal stages of activation. There was also some evidence for precocious sperm, but not egg, nuclear development when fertilization occurred in caffeine-containing medium.

Animals

Rate of fertilization in vitro and subsequent nuclear development as a function of the post-ovulatory age of the mouse egg.

Evidence is presented to indicate that older mouse eggs are fertilized more rapidly and also develop more rapidly to the pronuclear stage than younger ones. After a low (1.5 i.u.) or a high (7.5 i.u.) dose of PMSG, female mice were killed 13 or 17 h after hCG. The eggs were mixed in vitro with preincubated spermatozoa and fixed 1--1 1/4 h later. Although fertilization levels were high in all groups, the stages of egg activation and sperm head decondensation differed significantly. The observed ranking, from most to least rapid fertilization, of eggs obtained from females treated with 7.5 i.u. and killed 17 h after hCG, 1.5 i.u. and 13 h, and 7.5 i.u. and 13 h, was consistent with the approximate length of time the eggs had resided in the oviduct, i.e. the longer that time period, the more rapid the fertilization. When eggs were fixed 4 1/4 h after mixing with spermatozoa, the majority of older eggs were fully pronuclear while only a few of the younger eggs were as advanced, indicating accelerated nuclear development in the cytoplasm of the older eggs.

Animals

Separation of viable Rickettsia typhi from yolk sac and L cell host components by renografin density gradient centrifugation.

Rickettsia typhi cultivated in the yolk sac of chicken embryos or in L cells irradiated 7 days previously was separated from host cell components by two cycles of Renografin density gradient centrifugation. Preliminary steps involved differential centrifugation and centrifugation over a layer of 10% bovine plasma albumin of infected yolk sac suspensions, or trypsinization and passage through filters of wide porosity of infected L cell suspensions. Rickettsial preparations obtained by these methods appeared to be free from host cell components while retaining high levels of hemolytic activity, egg infectivity, and capacity to catabolize glutamate. Average yields were 3.3 mg of rickettsial protein per yolk sac or 0.44 mg per 16-oz (ca. 475-ml) L cell culture. Extracts from these two preparations displayed malate dehydrogenase activity of electrophoretic mobility identical to each other but quite different in migration patterns from the corresponding host cell enzymes. This method of separation of rickettsiae from host cell constituents appears to be particularly well suited for the study of rickettsial enzymatic activity.

Animals

The dynamics of maternal poly(A)-containing mRNA in fertilized sea urchin eggs.

Cytoplasmic polyadenylated RNA with the characteristics of sequestered mRNA exists in the unfertilized sea urchin egg. Following egg activation, the amount of poly(A) doubles, but total RNA content stays constant. Chromatography of the RNA on poly(U)-Sepharose shows that the amount of RNA that bears a poly(A) tract increases slightly (approximately 20-30%) during the 2 hr after fertilization. When a cDNA transcript of the poly(A)+ mRNA from 2 hr zygotes is reacted against poly(A)+ RNA from either eggs or zygotes, the kinetics of reassociation of the two preparations seem identical; hence the RNA sequences bearing poly(A) are the same in eggs and zygotes. Measurement of the length of the poly(A) tract in eggs and zygotes shows an increase in number average length from about 45 bases to 60 bases. Measurement of tract length of poly(A) in two cell zygotes by adenosine/AMP ratios of radioactive RNA shows that the poly(A) tract of the zygote is solely accounted for radioactive RNA, indicating extensive turnover of the poly(A). It is concluded that the poly(A) tract in these cells is subject to both lengthening and shortening, with the former predominating in this instance. the increase in poly(A) does not involve polyadenylation of different sequences, but is due to an increase in the number of polyadenylated sequences and the length of the poly(A) tracts that they bear.

Animals

Effect of dietary DL-ethionine on egg laying and activities of cytoplasmic and mitochondrial L-glycerol 3-phosphate dehydrogenases in liver and kidney of Japanese quail, Coturnix coturnix japonica.

The effect of dietary DL-ethionine on egg laying and activities of succinate dehydrogenases, cytoplasmic and mitochondrial L-glycerol 3-phosphate dehydrogenases, in liver and kidney of Japanese quail, Coturnix coturnix japonica was investigated. When quail at full laying were fed a diet supplemented with DL-ethionine at 0.05%, 0.10%, 0.15% and 0.30% (w/w), respectively, apparent DL-ethionine concentration dependent inhibitions of egg laying rate, weights of ovary and oviduct, and egg weight were observed. At the 0.30% level, activity per g tissue of cytoplasmic L-glygerol 3-phosphate dehydrogenase from liver and kidney decreased to about one-half of the initial activity. A significant difference in the activity of mitochondrial L-glycerol 3-phosphate and succinate dehydrogenase from liver and kidney was obtained between the control and the DL-ethionine fed quail, respectively. These results indicated that dietary DL-ethionine inhibits egg laying and activities of L-glycerol 3-phosphate dehydrogenases and succinate dehydrogenase of both liver and kidney of Japanese quail.

Animals

Activation of hamster eggs by pricking.

When mature unfertilized eggs of the golden hamster were picked with a glass needle, nearly 80% of them were activated. The diameter of needle was of critical importance for the successful induction of activation. It appeared that extracellular calcium was not necessary for the establishment (reception) of stimulation by pricking but was necessary for later phases of activation. The possible mechanisms by which pricking with a needle triggers the activation of the egg were compared with the mechanisms by which divalent ionophore induces activation.

Animals

Bacteriuria in urinary schistosomiasis in Egypt a prevalence survey.

An epidemiologic survey to assess the prevalence of bacteriuria in urinary schistosomiasis was carried out in a region endemic for urinary schistosomiasis in Egypt. Twenty of 390 (5.1%) school boys aged 5--16 years were bacteriuric. This prevalence rate is more than 10 times greater than that found in comparable surveys in areas non-endemic for urinary schistosomiasis. In this endemic population bacteriuria was found in 6.5% of active egg excreters and 2.3% of non-egg excreters.

Adolescent

[Long-term reproduction of triploid and tetraploid parthenogenetic clones of silkworms during artificial thermal parthenogenesis].

The triploid and tetraploid clones of the silkworm (Bombux mori L.) are capable to be reproduced for a long time by means of thermal parthenogenesis. The activated eggs are pigmented in 80-99%. The hatching of larvae in triploid clones amounts to 20% and in tetraploid ones to 25-30%. The pigmentation of eggs and hatching of larvae greatly vary in different years: 47 to 100% and 0 to 73%, resp. In this respect, the polyploid clones are similar to those diploid ones which are characterized by the low percentage of complete parthenogenesis. The causes for the variability of parthenogenesis indices in the polyploid clones are discussed.

Bombyx

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