Glutathione transferase activity during Bufo bufo development

High levels of glutathione transferase activity were measured during the development of the embryos of Bufo bufo including unfertilized eggs. After stage 4 glutathione transferase activity gradually decreased until stage 25 when the minimum was reached. No change in the number of isozymes was noted during development according to isoelectric focusing analysis performed on the cytosolic fractions of selected stages.     

Late hexokinase activity expression during Bufo bufo development

Hexokinase activity was detected in cytosols and homogenates from different developmental stages of Bufo bufo embryos starting from stage 17. Free glucose was measured in the embryo cytosol and was detected at each stage tested. At stage 15, a large increase of glucose content of the embryo cytosol occurs. Hexokinase expression in the embryo thus occurs after the increase of cytosol glucose content occuring at stage 15. The findings rule out that glucose by itself is the hexokinase inducer in vivo. The very low glucose utilization found by many authors during early amphibian development may be related to the late hexokinase expression during Bufo bufo development.     

Glutathione peroxidases and glutathione reductase activities during Bufo bufo development

Glutathione peroxidases and glutathione reductase activities are expressed from the early stage of Bufo bufo development. Selenium-dependent and selenium-independent glutathione peroxidase activities fluctuated independently. The activity of selenium-independent was found to be higher than that of selenium-dependent glutathione peroxidase through all stages of development. Glutathione reductase activity, after a slight fall from stage 4 to stage 7, constantly increased up to stage 25.     

Late hexokinase activity expression during Bufo bufo development.

Hexokinase activity was detected in cytosols and homogenates from different developmental stages of Bufo bufo embryos starting from stage 17. Free glucose was measured in the embryo cytosol and was detected at each stage tested. At stage 15, a large increase of glucose content of the embryo cytosol occurs. Hexokinase expression in the embryo thus occurs after the increase of cytosol glucose content occurring at stage 15. The findings rule out that glucose by itself is the hexokinase inducer in vivo. The very low glucose utilization found by many authors during early amphibian development may be related to the late hexokinase expression during Bufo bufo development.     

Keratin expression during early embryonic development of Bufo bufo gargarizans

Three anti-keratin MAbs were used to identifykeratins expressed in early embryos of Bufo bufogargarizans.MAb AF5 recognized three polypeptides ofkeratin in oocytes,fertilized eggs,up to neurula withMr of 68,65 and 60Kd respectively.At tailbud stage,three other keratins(62,58 and 54Kd)began to expressand could be detected by AF5.MAbs D10 and K12 gavedifferent results,both of them could identify four keratin-like molecules with unusual molecular weights(Mr 98,95,30 and 27 Kd).Moreover,D10 could also detect a 54 Kdkeratin in neurula and tailbud stage embryos,while K12could reveal,beside 54Kd keratin,other four more kera-tins(68,65,62 and 60 Kd).The possible interpretation ofthese results and their implications are discussed.     

Transcriptional analysis of tyrosinase gene expression during Bufo bufo development

Tyrosinase (EC.1.14.18.1.) is a widespread enzyme, in the phylogenetic scale, that produces melanin, from bacteria to man, by using as substrates monophenols, o-diphenols and molecular oxygen. In this work we have confirmed and demonstrated that during Bufo bufo development tyrosinase activity and gene expression first occur at developmental stages 17–18 (tail bud-muscular response) as detected by a spectrophotometric assay and qRT-PCR. As expected, also during B. bufo development tyrosinase gene is expressed after the late gastrula (stage 12), differently from Rana pipiens development when tyrosinase mRNA appears at the neural plate stage and enzyme activity at stage 20 (gill circulation). We have cloned and sequenced the B. bufo tyrosinase cDNA in order to prepare B. bufo tyrosinase cDNA specific primers (forward and reverse). Tyrosinase mRNA cloning has been performed by using degenerate primers prepared according to the anuran tyrosinase gene sequence coding for the copper binding sites. The expressions of tyrosinase gene and enzymatic activity during B. bufo development support that until the developmental stage 17, embryo melanin is of maternal origin and at this stage can start embryo melanin synthesis. A correlation exists between tyrosinase expression and O₂ consumption during B. bufo development.     

Physiological differentiation of the mitochondria during Bufo bufo development.

1) The oxygen consumption increases during Bufo bufo development in accordance with the two steps which border at the "heart beat" stage. 2) Cytochrome c oxidase activity is not proportional to the oxygen consumption: it is notable and constant in the first step, and it only increases in the second. 3) In the mitochondria of preneural embryos, citrate synthase, NADP+ dependent isocitrate dehydrogenase, and succinate dehydrogenase activities are very low in respect to malate dehydrogenase and glutamate oxaloacetate transaminase activities. The Krebs cycle results lowered at the condensing reaction level with acetyl accumulation when pyruvate is available. The same behavior has been observed in the Xenopus laevis oocytes and differentiated tissues. 4) The presence of a phosphagen system which is different from creatine phosphate and arginine phosphate, supporting ATP level, has been demonstrated in B. bufo embryos. 5) Mitochondria of postneural embryos are able to accomplish a complete Krebs cycle by increasing citrate synthase, and succinate dehydrogenase activities. 6) In all B. bufo development, malate dehydrogenase and glutamate oxaloacetate transaminase constitute a multienzymatic system by which the mitochondria accomplish a decarboxylic amino acid shunt required for the transformation of deutoplasm into protoplasm. This shunt is also operative in the X. laevis oocytes. 7) Through pyruvate production, by oxidative decarboxylation of malate, the NAD(P)+ dependent malic enzyme could carry out a fundamental anaplerotic function in the mitochondria which is specialized in the production of biosynthetic blocks belonging to the embryo in which the carbohydrates metabolism rather than the glycolytic activity is designed for pentose phosphate and glycerol phosphate synthesis for protein and cytomembrane production. 8) Consistent metabolic differences have been highlighted between B. bufo embryos and X. laevis embryos.     

Changes of a high molecular mass proteinase activity during Bufo bufo development

1. A chymotrypsin-like proteolytic activity was found both in unfertilized eggs and in embryos of Bufo bufo. 2. A dramatical change of activity can be observed in the course of embryonic development. The activity rapidly increases after fertilization up to the stage 9 followed by a fall to a level close to unfertilized eggs. 3. Gel chromatography analysis reveals, in all stages of development, the presence of a single peak of proteinase activity characterized by a very high molecular mass. 4. Proteinase activity, found change during the development of Bufo bufo, was characterized by substrate specificity, protease inhibitor and pH effect. All results obtained suggest that the chymotrypsin-like activity can be assigned to the multicatalytic proteinase.     

Utilization of yolk platelets during early embryonic development of Rana temporaria and Bufo bufo

Utilization of yolk platelets in cleaving embryos of Rana temporaria and Bufo bufo was studied by different methods. Morphological observations of yolk platelets of R. temporaria embryos at tail bud stage by transmission electron microscopy indicated four initial phases of platelet degradation. The pattern of these events is similar to that found in embryos of B. bufo. The morphological observations were confirmed by energy-dispersive X-ray microanalysis of the elemental content of platelets and by selected-area electron diffraction of platelet cores. Covalently bound sulphur content decreased during cleavage and the content of different inorganic ions changed, whereas the structure of crystalline core remained constant. Morphological changes found in the amorphous cortex of yolk platelets were due to their utilization. Stereological measurements indicated that utilization during cleavage increased, but only the initial phases of yolk platelet degradation were seen. The volume of the cortex did not decrease and the crystalline core did not fragment.     

Mitochondrial monoamine oxidase activity during preneural developmental stages of Bufo bufo

Monoamine oxidase specific activities against PEA and 5-HT have been measured in mitochondria isolated from early embryos of Bufo bufo. During the early development up to the neural fold stage, MAO activity undergoes a continuous decrease that is more evident when PEA is used as the substrate. The inhibition patterns of deprenyl and clorgyline demonstrate that, at the neural fold stage, both type A and B MAO are present. Both in eggs and embryos MAO type A activity appears slightly more sensitive to the inhibitory effect of various concentrations (0.1-2 M) of the denaturing agent urea.     

Behavioral reactions of Bufo bufo L. tadpoles to chemical excretion of adult toads and changes during larval development

The reactions of toad (Bufo bufo L.) tadpoles to chemical excretes of adults were studied. In double-choice test aquarium tadpoles demonstrated avoidance reactions (swimming away from the place of stimulus delivering, reduction time of spent in stimulus part of aquarium and reduction of movement activity). Efficiency of stimulus reduced when its concentration decreased. The thresholds of tadpoles sensitivity estimated according to the reduction of time spent in stimulus part of aquarium decreased in onthogeny: they were near 500 x 10(-5) g hour/l at 36-39 developmental stages and 500 x 10(-4) g.hour/l at 40-41 stages.     

Specific activities of 6-phosphogluconate dehydrogenase,glucose-6-phosphate dehydrogenase and glucose-6-phosphate isomerase during Bufo bufo development

It has been suggested by some authors that during amphibian development, due to the higher glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activity compared to that of 6-phosphogluconate dehydrogenase (EC 1.1.1.43), 6-phosphogluconate could accumulate in the embryo tissues and regulate the channelling of glucose-6-phosphate into glycolysis. Here, on the base of the specific activities of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glucose-6-phosphate isomerase (EC 5.3.1.9) found in the embryos of Bufo bufo during development, it is discussed whether 6-phosphogluconate can accumulate and play a regulative role on glucose-6-phosphate metabolism in the anuran embryo.     

中华大蟾蜍卵母细胞成熟过程中膜电位变化的实验分析

The full-grown oocytes obtained from toad (bufo bufo gargarizans) submitted in hibernation state or reared at 25-30 degrees C for several months, named hibernation oocyte or high temperature oocyte, had a membrane potential of -41.51 +/- 0.77 mV and -43.83 +/- 1.39 mV in Ringer's solution respectively. The hibernation oocytes underwent GVBD (germinal vesicle breakdown) and membrane depolarization at 19 +/- 1 degree C after progesterone stimulation. The membrane potential was about -20 mV at the period of GVBD, and -10 mV or so at 20 hours after the hormone treatment. However, the high temperature oocytes did not undergo GVBD, their membrane potential decreased before the fourth hour after treatment with progesterone and then recovered. If the hibernation oocytes were preincubated at 37-38 degrees C for 13 hours prior to the culture in the medium containing progesterone (10(-6)M, 37-38 degrees C), no GVBD was observed and the membrane depolarized before the fourth hour after treatment with progesterone then recovered, but MPF was detectable in the cytoplasm (unpublished). Both GVBD and membrane depolarization appeared in the hibernation oocytes and high temperature oocytes after injection of MPF. The time required for the hibernation oocytes injected MPF to attain the membrane potential about -20 mV was 4 hours earlier than that of progesterone treatment. It was just the time required for the appearance of MPF in the cytoplasm of oocytes treated with the hormone. It was noticed in our precedent article that a factor which appeared in the cytoplasm of high temperature oocytes differed from MPF. The factor was called Hibernation Oocyte Mature Promoting Factor (HOMPF).(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereological analysis of mitochondria in embryos of Rana temporaria and Bufo bufo during cleavage

Total numbers of mitochondria and their morphology have been quantitatively determined in mature oocytes and in cleaving embryos of two anuran species Rana temporaria and Bufo bufo using stereological methods. Surface densities of inner mitochondrial membranes for both studied species during cleavage ranged from 5.43 m2/cm3 to 7.53 m2/cm3, whereas volume densities of mitochondria did not exceed 1.65%. Since values of these parameters were low, thus embryos during cleavage may be considered as metabolically "silent". Transition of ultrastructural morphology of mitochondria towards that characterising actively respiring organelles occurs at stage 9 for R. temporaria and at stage 8 for B. bufo, correlated with blastula-gastrula and mid-blastula transition, respectively. The total numbers of mitochondria N(c) in mature oocytes are as high as 114.8 and 107.2 millions for R. temporaria and B. bufo, respectively, and during cleavage at late blastula stages they increase to 300 millions for both species under study. We suggest that an undefined mechanism might eliminate during cleavage those amphibian embryos which contain small number of mitochondria and low levels of nutrient substances.