Metabolic behaviour and cleavage capacity in the amphibian egg.
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During the winter season the full grown Bufo arenarum oocyte shows the metabolic behaviour characteristic of differentiated tissues of the same species. Due to seasonal variations, during the amplexus period, it acquires the metabolic behaviour of the segmenting egg. Short-time-induced ovulations (5-6 h) determine germinal vesicle breakdown immediately before the expulsion of the oocyte, without modifying the ovarian metabolism of the same. The incidence of the operative type of metabolism upon their capacity to cleave after insemination and needle pricking, has been studied in coelomic oocytes, which have attained nuclear maturation and have not experienced oviducal secretion effects. The results obtained indicate that the segmenting capacity of the egg is attained only when, through biochemical modifications, the oocyte acquires the metabolic behaviour characterizing embryonic cells. It is postulated that the metabolic changes observed in the oocyte constitue a fundamental aspect of cytoplasmic maturation. (+info)
Vitelline envelope of Bufo arenarum: biochemical and biological characterization.
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Vitelline envelopes (VEs) of Bufo arenarum were isolated in order to study their composition and their role in fertilization. VEs are composed of four glycoproteins, with molecular masses of 120, 75, 41, and 38 kDa. To characterize its biological properties, we quantitatively determined sperm-VE binding and the induction of the acrosome reaction. Heterologous binding of B. arenarum sperm to Xenopus laevis VE components was observed with about one-third the efficiency of homologous binding. Equivalent binding of X. laevis sperm to the B. arenarum VE was observed. When B. arenarum sperm were incubated with fluorescein isothiocyanate-labeled VE, the labeled glycoproteins bound to the anterior end of the sperm head, showing a lateral distribution. Induction of the acrosome reaction was evaluated by incubating sperm in hypotonic saline media with VE glycoproteins. VEs induced the acrosome reaction in a time- and concentration-dependent manner. The acrosome reaction was maximal after 10 min. The half-maximal effect was obtained at a glycoprotein concentration of 1 microg/ml. Specificity was determined using fertilization envelope glycoproteins, which failed to induce the acrosome reaction. The B. arenarum VE is biochemically similar to other egg envelopes. It also seems that its biological properties are similar to other species in regard to sperm binding and induction of the acrosome reaction. However, as far as we are aware, this is the first observation of the VE inducing the sperm acrosome reaction in amphibians. The relatively small differences observed in heterologous sperm-VE binding in X. laevis and B. arenarum are inconsistent with the current paradigm that species specificity in fertilization is regulated at the sperm-VE binding step. (+info)
The envelopes of amphibian oocytes: physiological modifications in Bufo arenarum.
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A characterization of the Amphibian Bufo arenarum oocyte envelope is presented. It was made in different functional conditions of the oocyte: 1) when it has been released into the coelomic cavity during ovulation (surrounded by the coelomic envelope, (CE), 2) after it has passed through the oviduct and is deposed (surrounded by the viteline envelope, (VE), and 3) after oocyte activation (surrounded by the fertilization envelope, (FE). The characterization was made by SDS-PAGE followed by staining for protein and glycoproteins. Labeled lectins were used to identify glycosidic residues both in separated components on nitrocellulose membranes or in intact oocytes and embryos. Proteolytic properties of the content of the cortical granules were also analyzed. After SDS-PAGE of CE and VE, a different protein pattern was observed. This is probably due to the activity of a protease present in the pars recta of the oviduct. Comparison of the SDS-PAGE pattern of VE and FE showed a different mobility for one of the glycoproteins, gp75. VE and FE proved to have different sugar residues in their oligosaccharide chains. Mannose residues are only present in gp120 of the three envelopes. N-acetyl-galactosamine residues are present in all of the components, except for gp69 in the FE. Galactose residues are present mainly in gp120 of FE. Lectin-binding assays indicate the presence of glucosamine, galactose and N-acetyl galactosamine residues and the absence (or non-availability) of N-acetyl-glucosamine or fucose residues on the envelopes surface. The cortical granule product (CGP) shows proteolytic activity on gp75 of the VE. (+info)
The effect of UV-B radiation on Bufo arenarum embryos survival and superoxide dismutase activity.
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The exposure of Bufo arenarum embryos to 300-310 nm UV-B at a dose of 4,104 Joule/m(2) resulted in 100% lethality within 24 hr while 820 Joule/m(2) was the NOEC value for short-term chronic (10 days) exposure. The dose response curves show that lethal effects are proportional with the dose and achieve its highest value within 48 hr post exposure. The superoxide dismutase (SOD) activity in amphibian embryos for sublethal UV-B exposures was evaluated by means of UV-B treatments with 273 (A), 820(B), 1368(C) and 1915(D) Joule/m(2) at 2 and 5 hours post irradiation. The SOD activity in units/mg protein in A, B, C and D at 2 hr after treatments were 80.72 +/- 14.29, 74.5 +/- 13.19, 39.5 +/- 6.99 and 10.7 +/- 1.89 respectively while for control embryos it was 10.88 +/- 1.31. At 5 hr after treatments the SOD values were similar to those found in control embryos. The results confirm the high susceptibility of amphibian embryos to UV-B and point out that the SOD activity is enhanced by low doses of UV-B irradiation achieving significantly higher values than in control embryos at 2 hr post exposure. (+info)
Synergistic effects of copper and butylic ester of 2,4-Dichlorophenoxyacetic acid (Esternon Ultra) on amphibian embryos.
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Cu2+ and butylic ester of 2,4-Dichlorophenoxyacetic acid as Esternon Ultra (2,4-D) toxicity on Bufo arenarum embryos were evaluated by means of a short-term chronic toxicity test (AMPHITOX). The NOEC values for Cu and 2,4-D were 0.02 mg/L and 2 mg/L respectively. The toxicity profile curves for Cu and 2,4-D were reported. The interactions of the metal and the herbicide were evaluated by combined treatments with different concentrations of Cu and 2,4-D. Although in all cases, a synergistic effect between these chemicals was observed, the combination of concentrations exerting low level effects in isolated treatments resulted in more adverse embryonic survival. Considering that both products are extensively used in agroecosystems, this fact could be of concern for non target species like amphibians. (+info)
Egg water from the amphibian Bufo arenarum induces capacitation-like changes in homologous spermatozoa.
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Mammalian sperm acquire fertilizing capacity after residing in the female tract, where physiological changes named capacitation take place. In animals with external fertilization as amphibians, gamete interactions are first established between sperm and molecules of the egg jelly coat released into the medium. Since dejellied oocytes are not normally fertilized, the aim of this study was to determine if the jelly coat of the toad Bufo arenarum promotes a "capacitating" activity on homologous sperm. We found that sperm incubation in diffusible substances of the jelly coat (egg water) for 90-180 s is sufficient to render sperm transiently capable of fertilizing dejellied oocytes. The fertilizing state was correlated with an increase of protein tyrosine phosphorylation and a decrease of sperm cholesterol content. Inhibition of either the increase in tyrosine phosphorylation or cholesterol efflux affected the acquisition of fertilizing capacity. Phosphorylation and fertilization could be promoted with NaHCO(3) and also by addition of beta cyclodextrin. Moreover, sperm could gain the ability to fertilize dejellied oocytes in the presence of these compounds. These data indicate that sperm should undergo a series of molecular changes to gain fertilizing capacity; these changes are reminiscent of mammalian sperm capacitation and take place before the acrosome reaction. (+info)
Acclimation to low level exposure of copper in Bufo arenarum embryos: linkage of effects to tissue residues.
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The acclimation possibilities to copper in Bufo arenarum embryos was evaluated by means of three different low level copper exposure conditions during 14 days. By the end of the acclimation period the copper content in control embryos was 1.04 +/- 0.09 microg g(-1) (wet weight) while in all the acclimated embryos a reduction of about 25% of copper was found. Thus copper content could be considered as a biomarker of low level exposure conditions. Batches of 10 embryos (by triplicate) from each acclimation condition were challenged with three different toxic concentrations of copper. As a general pattern, the acclimation protocol to copper exerted a transient beneficial effect on the survival of the Bufo arenarum embryos. The acclimation phenomenon could be related to the selection of pollution tolerant organisms within an adaptive process and therefore the persistence of information within an ecological system following a toxicological stressor. (+info)
Inhibition of Bufo arenarum oocyte maturation induced by cholesterol depletion by methyl-beta-cyclodextrin. Role of low-density caveolae-like membranes.
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