Pyruvate kinase isozymes in oocytes and embryos from the frog Xenopus laevis

1. The kinetic characteristics of pyruvate kinase isozymes from oocytes, embryos, liver and skeletal muscle from the clawed frog Xenopus laevis were measured in cell extracts. 2. The muscle and liver isozymes display Michaelis-Menten kinetics with Kms for phosphoenolpyruvate (PEP) of 0.02 and 0.05 mM, respectively. 3. Pyruvate kinase from oocytes and embryos displays cooperative kinetics for PEP with a Km of about 0.15 mM; the kinetics become hyperbolic and the Km for PEP is reduced to 0.05 mM in the presence of microM concentrations of fructose-1,6-bisphosphate. 4. These data serve to characterize pyruvate kinase activity in oocytes and embryos and the kinetics are compared to mammalian pyruvate kinase isozymes.     

Accumulation of free amino acids in growing Xenopus laevis oocytes

The sizes of amino acid pools in growing Xenopus laevis oocytes have been measured. The total free amino acid content per oocyte increases nearly 25-fold during oocyte growth. Together, glutamic acid and aspartic acid account for approximately 59-75% of the total amino acid pool in Xenopus oocytes. On the other hand, methionine and cysteine are the least abundant of the amino acids detected, each accounting for less than 0.7% of the total pool in developing oocytes. It is argued that the acid-extractable amino acid pool represents the precursor pool used in protein synthesis.     

The degradation of ribonucleic acids injected into Xenopus laevis oocytes

Different radioactive RNAs were injected into Xenopus laevis oocytes, and their degradation followed with time. Deproteinized ribosomal RNAs and synthetic polynucleotides, with the exception of polyadenylic acid, were degraded rapidly with apparent first order kinetics and half-lives ranging from 1–6 hr. Transfer RNA, poly(A), and ribosomal RNA injected as whole ribosomal particles were quite stable during the period studied (20 hr). Messenger RNAs from Dictyostelium discoideum and Vesicular Stomatitis Virus, which have poly(A) sequences at their 3′ terminus, presented biphasic degradation kinetics. Approximately 60% of these RNAs was degraded in the first 6 hr, whereas the remaining 30–40% was stable for at least 22 hr. Analysis of the stable material by sucrose gradients showed that it had the same sedimentation pattern as the original material, except that it contained, in addition, free poly(A) sequences sedimenting somewhat smaller than 4S. Puromycin treatment of the cells injected with Dictyostelium mRNAs reduced the percentage of stable RNA to 10%, approximately the poly(A) content of these RNAs. Similar treatment with emetine, which also inhibited cellular protein synthesis, did not affect the stable mRNA fraction.     

The metabolism of ribosomal proteins microinjected into the oocytes of Xenopus laevis

When the total proteins from Xenopus laevis 60 S ribosomal subunits (TP60) were 3H-labeled in vitro and injected back into X. laevis oocytes, most 3H-TP60 are integrated into the cytoplasmic 60 S subunits via the nucleus during 16 h of incubation. In the oocytes whose rRNA synthesis is inhibited, 3H-TP60 are rapidly degraded with a half-life of 2-3 h. This degradation ceased as soon as rRNA synthesis was resumed, suggesting that ribosomal proteins unassociated with nascent rRNA are unstable in the oocytes. The degradation of 3H-TP60 in the absence of RNA synthesis was inhibited by iodoacetamide, a cysteine protease inhibitor, resulting in the accumulation of 3H-TP60 in the nucleus reaching about a threefold concentration in the cytoplasm. Considering the results with enucleated oocytes, we suggest that the X. laevis nucleus has a limited capacity to accumulate ribosomal proteins in an active manner but that those ribosomal proteins accumulated in excess over rRNA synthesis are degraded by a cysteine protease in the nucleus. By contrast, ribosomal proteins from Escherichia coli only equilibrate between the nucleus and the cytoplasm and are degraded by serine protease(s) in the cytoplasm without being integrated in the form of ribosomes in the nucleus.     

Transport of amino acids and nucleosides in metaphase-arrested unfertilized oocytes of Xenopus laevis

Metaphase-arrested, unfertilized shed oocytes of Xenopus laevis obtained after hormonal stimulation of the female are able to take up nucleosides (U, T) and amino acids (Ala, Gly, Glu, Gln, Tyr). For alanine, tyrosine, and glutamic acid the transport is uphill. The transport of the amino acids studied is activated by Na+, whereas the uptake of the nucleosides is independent of the Na+ concentration. Ouabain does not inhibit the uptake of amino acids significantly. The uptake of alanine and thymidine is not measurably affected by the presence of the jelly coat.     

Long-chain polyunsaturated fatty acids and the regulation of bone metabolism

The role of prostaglandin E2 (PGE2) in the regulation of bone remodeling is well established. There is increasing evidence that various long-chain polyunsaturated fatty acids (LCPUFAs), as well as nonprostanoid LCPUFA metabolites, also have critical roles in regulating bone metabolism and may have therapeutic potential in the management of postmenopausal osteoporosis. Although only the 18-carbon precursors for the n-3 and n-6 LCPUFAs are deemed "dietary essential," the ability of the body to convert these precursor fatty acids into the more highly unsaturated 20- and 22-carbon LCPUFAs decreases with aging, menopause, and various lifestyle factors (e.g., smoking). Increasing dietary LCPUFA intake increases tissue and blood LCPUFA concentrations, as well as the concentrations of their metabolites. Modification of dietary LCPUFA content, particularly increasing the intake of n-3 LCPUFAs, has been shown to minimize the decline in bone mass caused by menopause in women and ovariectomy in animal models. This review summarizes findings from both in vivo and in vitro studies and outlines the effects of LCPUFAs and their metabolites on calcium balance, osteoblastogenesis, osteoclastogenesis, and osteoblast and osteoclast function.     

Nephroblastoma in the clawed frog Xenopus laevis.

One 2.5-3 year old female clawed frog (Xenopus laevis), out of a consignment of 4,000 frogs, was found to have an abnormal abdominal growth, weighing 7.9 g. The growth was examined histologically and on the basis of the abundant stroma and serially-arranged tubules nephroblastoma was diagnosed. The growth is not considered to be transmissible.     

Polyadenylic acid-containing RNA in Xenopus laevis oocytes

The quantity of poly(A)-containing RNA is measured in Xenopus laevis oocytes as a function of developmental stage. The amount of poly(A)-containing RNA per oocyte, 0.7 to 1.0% of the total RNA, remains relatively constant from early vitellogenesis until ovulation. It is largely present in the cytoplasm of the oocyte in the form of a ribonucleoprotein complex. The poly(A) sequence is approximately 100 bases in length and is attached to molecules of heterogeneous sedimentation coefficients.     

Induction of maturation in small Xenopus laevis oocytes

The competence of Xenopus laevis oocytes in various stages of growth to respond to progesterone treatment was investigated. Full-grown (stage 6) oocytes undergo nuclear membrane dissolution and resume meiosis in response to progesterone exposure, while smaller oocytes (stages 3-5; less than 1100 micron in diameter) do not. The defect which prevents 750- to 1050-micron oocytes from responding to progesterone can be overcome by microinjecting cytoplasm withdrawn from a stage 6 oocyte. Germinal vesicle breakdown in these small oocytes occurs on a timetable similar to that of stage 6 oocytes exposed to progesterone and is accompanied by a twofold increase in protein synthesis as well as the activation of MPF. The results argue that a cytoplasmic factor(s) which probably first appears at late stage 5 is required for progesterone responsiveness. The identity and role of the factor(s) in the development of maturation competence and the regulation of maternal mRNA translation are discussed.     

Mitochondria DNA synthesis in oocytes of Xenopus laevis

The process of attachment was studied in primary mouse kidney epithelial cell cultures by means of reflexion contrast microscopy, a method developed for studying the cell membrane-substrate relationship. The first in a series of events is simple adherence to the substrate, called close contact. This phenomenon is associated with the greatest extension of lamellar cytoplasm and the fewest number of cell nuclei/unit area. The nuclei of such cells are in close contact with the bottom portion of the cell membrane. Approx. 24 h after planting, as the cultures become more crowded, cells develop a different kind of attachment to the substrate—focal contacts—that are correlated with a decrease in lamellar cytoplasm. Cells detached from the substrate after close contact formation readily reattach, while cells detached after formation of focal contacts do not reattach. After incubation for periods greater than 5 days, the dense cultures degenerate and cells lose their attachment to the glass surface.     

Rabies mRNA translation in Xenopus laevis oocytes.

Two rabies virus-specific mRNA species were identified by analysis of their encoded proteins after translation of the partially purified species in Xenopus laevis oocytes. One of these coded for the virion surface glycoprotein (G protein), and the other coded for the major structural protein of the virion nucleocapsid (N protein). The G-mRNA sedimented in a sucrose density gradient at about 18S, and the N-mRNA had a sedimentation coefficient of approximately 16S. Their respective translation products were identified in a radioimmunoassay with specific monoclonal antibody probes that recognized only G or N proteins. Immunoprecipitates formed between the radiolabeled viral antigens synthesized in programmed oocytes and their respective monoclonal antibodies were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The glycoprotein antigen translated from G-mRNA in oocytes migrated in the gel ahead of the virion G protein with a migration rate that was similar to that of nonglycosylated intracellular glycoproteins from virus-infected cells. The results suggested that the branched-chain carbohydrate of G protein was not required for recognition by the particular monoclonal antibody used. The nucleocapsid antigen translated from N-mRNA in oocytes migrated to the same position in the gel as marker virion N protein. Both the electrophoretic mobility of virus-specific antigens in sodium dodecyl sulfate-polyacrylamide gel and the antibody concentration dependence for immunoprecipitations were criteria for identifying the individual viral proteins encoded by the two rabies mRNA's.     

Soluble cytokeratins in Xenopus laevis oocytes and eggs

Xenopus oocytes contain a radial network of cytokeratins which seems to fragment during meiosis reinitiation (maturation). The mature egg contains only a cortical network of cytokeratins. We have looked for the presence of soluble cytokeratins in oocytes and unfertilized eggs and have found them in both cases. However, the proportion of soluble to insoluble cytokeratins is slightly higher in the egg than in the oocyte. Soluble cytokeratins incorporate 35S-methionine at a high rate in the oocyte but to a lesser extent in the egg. This suggests that they are biosynthetic intermediates in the oocyte. In the egg, at least a fraction of the soluble cytokeratins may arise from the fragmentation of the polymer which seems to occur during the maturation process. Insoluble cytokeratins are strongly labeled with 32P both in oocytes and eggs. On the other hand only the soluble keratins of the egg incorporate 32P. Since the isoelectric point of soluble and insoluble cytokeratins is the same in oocytes and eggs, their absolute level of phosphorylation probably remains relatively constant. This suggests that: i) phosphate turnover is very slow in oocyte soluble cytokeratins, ii) phosphorylation is not a major way of changing the structural state of cytokeratins in amphibian oocytes and eggs.     

An endogenous monocarboxylate transport in Xenopus laevis oocytes

We investigated the existence of an endogenous system for lactate transport in Xenopus laevis oocytes. (36)Cl-uptake studies excluded the involvement of a DIDS-sensitive anion antiporter as a possible pathway for lactate movement. L-[(14)C]lactate uptake was unaffected by superimposed pH gradients, stimulated by the presence of Na(+) in the incubating solution, and severely reduced by the monocarboxylate transporter inhibitor p-chloromercuribenzenesulphonate (pCMBS). Transport exhibited a broad cation specificity and was cis inhibited by other monocarboxylates, mostly by pyruvate. These results suggest that lactate uptake is mediated mainly by a transporter and that the preferred anion is pyruvate. [(14)C]pyruvate uptake exhibited the same pattern of functional properties evidenced for L-lactate. Kinetic parameters were calculated for both monocarboxylates, and a higher affinity for pyruvate was revealed. Various inhibitors of monocarboxylate transporters reduced significantly pyruvate uptake. These studies demonstrate that Xenopus laevis oocytes possess a monocarboxylate transport system that shares some functional features with the members of the mammalian monocarboxylate cotransporters family, but, in the meanwhile, exhibits some particular properties, mainly concerning cation specificity.     

Amino acid uptake in Xenopus laevis oocytes.

The isolated oocytes from Xenopus laevis are able to take up radioactive amino acids from the exogenous medium. Most amino acids tested are taken up to reach concentrations higher than the extracellular medium. The initial uptake velocities vary with the external amino acid concentration in a Michaelis-Menten fashion. Aspartic acid requires concentrations an order of magnitude higher than the five other amino acids tested to reach half the maximal uptake velocity. The uptake mechanism seems to be specific for groups of analogous amino acids, as can be determined by competition studies. The amino acid groups for which there is some evidence of uptake specificity would be aromatic, aliphatic, acidic and basic. Amino acid pools of oocytes show that these cells can concentrate amino acids from Xenopus blood, as well as from artificial media.