In vivo synthesis and turnover of cytoplasmic ribosomal RNA by stage 6 oocytes of Xenopus laevis.

Cytoplasmic ribosomal RNA (rRNA) synthesis was detected in white-banded stage 6 oocytes taken from female Xenopus laevis which were injected with [³H]guanosine 7 days previously. The specific radioactivity of the rRNA in oocytes collected from injected females by weekly laparotomies displays first-order exponential decay. Calculated values for the half-life of rRNA ranged from 9.1–30.9 days in experiments on four animals. The concept of ribosomes in large ovarian oocytes of amphibians as an absolutely stable, long-term storage product appears incorrect.     

Quantitative and qualitative analysis of RNA synthesis in stage 6 and stage 4 oocytes of Xenopus laevis

RNA synthesis has been studied in oocytes taken from Xenopus laevis females which have not recently ovulated. Such females contain a population of large (stage 6) oocytes which exhibit white equatorial bands and which are considered to represent the terminal stage of oocyte development. Rates of RNA synthesis in these “banded” oocytes were measured by analyzing the kinetics of incorporation of ³H-guanosine into acid-precipitable, alkaline-labile material, and changes in precursor pool (GTP) specific activity during incubations. In additional experiments, rates of RNA synthesis were measured after ³H-GTP was injected directly into stage 6 oocytes. For comparison, rates of RNA synthesis were measured in lampbrush chromosome stage oocytes (stage 4; 0.5–0.6 mm diameter). The results show that, under the in vitro conditions employed, stage 6 oocytes are not metabolically dormant, but synthesize total RNA at a rate at least as great as the stage 4 oocytes.Qualitative studies on newly synthesized RNA in the two oocyte classes have been performed using sucrose density gradient centrifugation and acrylamide gel electrophoresis. Both stage 4 and stage 6 oocytes exhibited similar patterns, and the bulk of the RNA synthesized and accumulated during 12-hr pulses appears to be ribosomal. These observations are discussed in terms of existing concepts concerning synthetic activity in stage 6 oocytes.     

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.     

Cyclic AMP synthesis in Xenopus laevis oocytes: inhibition by progesterone.

[alpha-32P]ATP was microinjected into Xenopus oocyte and neosynthesized cyclic AMP was isolated. Cholera toxin inhibited progesterone-induced maturation and stimulated after 3 h of preincubation the amount of neosynthesized cyclic AMP. Progesterone decreased the neosynthesis of cyclic AMP during the first hour following addition of the hormone.     

Cyclic AMP synthesis in Xenopus laevis oocytes inhibition by progesterone

[α-³²P] ATP was microinjected into Xenopus oocyte and neosynthesized cyclic AMP was isolated. Cholera toxin inhibited progesterone-induced maturation and stimulated after 3 h of preincubation the amount of neosynthesized cyclic AMP. Progesterone decreased the neosynthesis of cyclic AMP during the first hour following addition of the hormone.     

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.     

Translation of avian sarcoma virus RNA in Xenopus laevis oocytes.

Evidence for the synthesis and processing of Pr76 (precursor to group-specific antigens p27, p19, and $\text{p12}\text{15}$, upon injection of avian sarcoma virus 70S or 35S RNA into $\text{Xenopus}$ oocytes has been presented. Further, we show that tRNA^trp primer, bound to 35S RNA, does not block translation of virion RNA under these conditions.     

Protein synthesis in oocytes of Xenopus laevis is not regulated by the supply of messenger RNA

The control of protein synthesis in oocytes of Xenopus laevis has been investigated by injecting oocytes with mRNA and polysomes followed by labeling with ¹⁴C-amino acid mixtures. Contrary to previous reports in which injected oocytes were labeled with ³H-histidine, injected globin mRNA is found to decrease amino acid incorporation into endogenous proteins competitively at all concentrations tested. No increase in overall amino acid incorporation is detected when more mRNA is supplied. Similar results are obtained after labeling injected oocytes with leucine, methionine, proline or valine individually. An explanation is presented for the conflicting results obtained when histidine is used as a label.When reticulocyte polysomes are injected, rather than purified globin mRNA, incorporation of amino acids into endogenous proteins remains roughly constant and overall incorporation increases. Similarly, when encephalomyocarditis viral RNA is injected together with either globin mRNA or reticulocyte polysomes, the globin mRNA causes decreased amino acid incorporation into encephalomyocarditis proteins, but the polysomes do not do so. The results demonstrate that different types of mRNA compete for a strictly limited translational capacity which is saturated in the normal oocyte. The limiting component is present in polysomes and is not message-specific. The constraint on protein synthesis in the amphibian oocyte cannot be fully explained by masked mRNA.     

Ectoenzymatic breakdown of diadenosine polyphosphates by Xenopus laevis oocytes.

Xenopus laevis oocytes exhibit ectoenzymatic activity able to hydrolytically cleave extracellular diadenosine polyphosphates (Ap(n)A). The basic properties of this ectoenzyme were investigated using as substrates di-(1,N(6)-ethenoadenosine) 5',5"'-P(1),P(4)-tetraphospate [epsilon-(Ap(4)A)] and di-(1,N(6)-ethenoadenosine) 5',5"'-P(1),P(5)-pentaphospate [epsilon-(Ap(5)A)], fluorogenic derivatives of Ap(4)A and Ap(5)A, respectively. epsilon-(Ap(4)A) and epsilon-(Ap(5)A) are hydrolysed by folliculated oocytes according to hyperbolic kinetics with K(m) values of 13.4 and 12.0 microM and Vmax values of 4.8 and 5.5 pmol per oocyte per min, respectively. The ectoenzyme is activated by Ca(2+) and Mg(2+), reaches maximal activity at pH 8--9 and is inhibited by suramin. Defolliculated oocytes also hydrolyse both substrates with similar K(m) values but V(max) values are approximately doubled with respect to folliculated controls. Chromatographic analysis indicates that extracellular epsilon-(Ap(4)A) and epsilon-(Ap(5)A) are first cleaved into 1,N(6)-ethenoAMP (epsilon-AMP) + 1,N(6)-ethenoATP (epsilon-ATP) and epsilon-AMP + 1,N(6)-ethenoadenosine tetraphosphate (epsilon-Ap(4)), respectively, which are catabolized to 1,N(6)-ethenoadenosine (epsilon-Ado) as the end product by folliculated oocytes. Denuded oocytes, however, show a drastically reduced rate of epsilon-Ado production, epsilon-AMP being the main end-product of extracellular epsilon-(Ap(n)A) catabolism. Results indicate that, whereas the Ap(n)A-cleaving ectoenzyme appears to be located mainly in the oocyte, ectoenzymes involved in the dephosphorylation of mononucleotide moieties are located mainly in the follicular cell layer.     

Correlation between ultrastructural aspect of nucleoli and inhibition of ribosomal RNA synthesis in Xenopus laevis oocytes

The structure of the several kinds of nucleoli successively present in growing Xenopus oocytes before vitellogenesis suggests an inhibition of ribosomal RNA synthesis during this period. Such an inhibition is in good agreement with the high content of low molecular weight RNA in these oocytes.     

An RNA molecule copurifies with RNase P activity from Xenopus laevis oocytes.

Utilizing a procedure for the purification of RNase P from Xenopus laevis germinal vesicle (GV) extracts, according to which the contamination by a large, cytoplasmic, cylindrical structure (1) is avoided, we demonstrate that the X.laevis enzyme, like the HeLa RNase P, is precipitated by anti-Th antibodies and an RNA molecule (XL RNA), 320 nucleotides long, copurifies with the activity. The sequence of XL RNA is 60% homologous to HeLa H1 RNA, therefore the two molecules seem related.     

Effect of ouabain on the meiotic maturation of stage IV-V Xenopus laevis oocytes

Full-grown stage VI Xenopus laevis oocytes (1,200 to 1,300 micron) respond to progesterone stimulation by undergoing a series of physiological and morphological changes that are referred to as meiotic maturation. Oocytes in earlier stages of oogenesis (I through V) do not undergo these changes and remain in prophase arrest when exposed to this steroid. We have found that oocytes ranging from 850 micron (stage IV) to 1,000 micron (stage V) are capable of responding to progesterone under the appropriate conditions. Oocytes greater than or equal to 850 micron in diameter underwent germinal vesicle breakdown (GVBD) after 10-12 hr of exposure to progesterone when ouabain was added to the medium at a concentration greater than 2.5 X 10(-6) M. Under this culture condition, progesterone was now able to induce a 0.3- to 0.4-unit increase in the intracellular pH of stage IV-V oocytes, a 4- to 5-fold increase in 40s ribosomal protein S-6 phosphorylation, and a 2.3-fold increase in their rate of protein synthesis. All of these physiological changes are characteristic of full-grown stage VI oocytes undergoing meiotic maturation. In addition, we have found that oocytes greater than or equal to 750 micron are capable of amplifying maturation promoting factor (MPF) in their cytoplasm leading to GVBD. Therefore, stage IV-V Xenopus oocytes have the potential for undergoing meiotic maturation, but they are blocked at a point in prophase that appears to be alleviated by the combination of progesterone and ouabain.     

The effect of cytochalasin D on protein synthesis in Xenopus laevis oocytes

Defolliculated fully grown oocytes of Xenopus laevis were treated with cytochalasin D (10 micrograms/ml) and their protein synthesis was studied by labelling with S-35 methionine. This treatment brought about an alteration in pigment pattern as well as a reduction in amino acid uptake by the oocytes. However, the radioactive amino acid taken by cytochalasin-treated oocytes was incorporated into protein in the same proportion as in untreated oocytes. These results suggested that subcortical pigment distribution and amino acid uptake in fully grown oocytes were microfilament-dependent processes, whereas protein synthesis in the oocyte was not.