Injection of an antibody against a p21 c-Ha-ras protein inhibits cleavage in axolotl eggs.

The presence of a ras protein was demonstrated in cleaving axolotl eggs by selective immunoprecipitation with a polyclonal antibody against a peptide encoded by the c-Ha-ras oncogene, cellular homolog of the v-Ha-ras oncogene of Harvey rat sarcoma virus. Injection of this antibody into axolotl oocytes subjected to progesterone treatment does not prevent meiotic maturation. Injection of the same antibody into a blastomere of axolotl eggs at the 2- or 4-cell stage causes cleavage arrest in the descendants of the injected blastomere. Cytological observations of the injected eggs show, in the arrested blastomeres, enlarged nuclei always surrounded by an intact nuclear envelope and containing uncondensed chromatin. The possible role of ras protein in meiosis and mitosis is discussed.

Bioluminescent assay of ATPase activity in embryonic material using firefly luciferase.

The continuous bioluminescent assay of ATP has been adapted to the study of Mg2+-dependent ATPases, including the (Na+; K+) pump, in amphibian tissues. A discrete bioluminescent assay procedure for ATPase has also been developed. Components of the firefly luciferase assay reagent modify the observed ATPase activity but this can be circumvented by performing discrete instead of continuous measurements of enzyme activity. In assays with commercial ATPase preparations the continuous bioluminescent assay procedure gave ATPase activities 2.2-fold lower than obtained with the discrete procedure. In Xenopus oocyte or egg homogenates, in contrast, the total ATPase activity measured is stimulated eight times by the luciferase reagent, mainly through an unexplained activation of a Mg2+-independent ATPase. In other tissues, such as Xenopus brain homogenates, both the continuous and discrete monitoring procedures are equally suitable for the determination of ATPase activity.

Cytological Effects of Heat-Shocks on Xenopus Oocytes and Eggs: (cytasters/spindles/furrows/nucleoli).

Heat-shocks (80 min at 34°c) induce the appearance of aster-like fibrous structures (cytasters) in maturing Xenopus oocytes. Cytaster formation is suppressed by treatments with colchicine or nocodazole of heat-shocked maturing oocytes. Heat-shocks destroy the meiotic spindle, but have no effect on cytasters induced by D2 O treatment. Heat-shocks (20 min at 36°c) also induce the formation of cytasters in unfertilized and freshly fertilized eggs of some females, but not in cleaving eggs. They suppress amphimixy, induce the regression of existing cleavage furrows and destroy the mitotic apparatuses in cleaving eggs. The arrested blastomeres often contain micronuclei (probably unfused telophase swollen chromosomes) where small basophilic nucleoli are frequently seen. The significance of these results is discussed.

Heat-shock response in Xenopus oocytes during meiotic maturation and activation.

After a 60 min heat-shock at 36 degrees C, Xenopus oocytes are still able to accomplish a complete meiotic maturation in response to a progesterone treatment. The 36 degrees C heat-shock applied to maturing oocytes strongly enhances the synthesis of a single heat-shock protein of approx. 70 000 molecular weight (hsp70); after activation with the Ca2+-ionophore A 23187, matured oocytes still display the ability to synthesize hsp70 and to survive a heat-shock. A cycloheximide treatment combined with a heat-shock induces, during the recovery period, the synthesis of two heat-shock proteins, of approx. 70 000 and 83 000 molecular weight.

[Phosphorylation of the ribosomal protein S6 during maturation of Xenopus oocytes: identification of the phosphorylated amino acids]. / Phosphorylation de la protéine ribosomiale S6 au cours de la maturation de l'oocyte de Xénope: identification des acides aminés phosphorylés.

The S6 protein from the small subunit of Xenopus oocytes in phosphorylated during progesterone-induced maturation. The identity of the amino acids phosphorylated in the S6 protein during this process has been established by two-dimensional electrophoresis followed by radioautography. The only phosphorylated amino acid we could detect is phosphoserine.

Ionic requirements for induction of maturation (meiosis) in full-grown and medium-sized Xenopus laevis oocytes.

There is already good evidence that calcium ions are involved in the induction of maturation in full-grown amphibian oocytes; we show here that other cations (K+, Mg2+) also play a role in this process. Full-grown (1.3 mm in diameter) and medium-sized (0.8-1.0 mm in diameter) oocytes were compared in the present study. It was found that, provided the medium is K+-free, valinomycin induces maturation in full-grown, but not in medium-sized, oocytes. Increasing the CaCl2 (20 mM) or the MgSO4 (40 mM) content of the medium induces maturation in full-grown, but not in medium-sized, oocytes; however, the latter undergo germinal vesicle breakdown after treatment with either progesterone or ionophore A23187 if there is an excess of Ca2+ or Mg2+ in the medium. Maturation is possible in a Na+-free medium, but amiloride inhibits germinal vesicle breakdown when NaCl is present in the medium. It is concluded that maturation is controlled by changes in the balance between the various ions rather than by Ca2+ alone.

[Induction of maturation (meiosis) in small oocytes of Xenopus. A cytological study]. / Induction de la maturation (méisose) dans de petits oocytes de xénope. Etude cytologique

Injection of endoplasm from large Xenopus oocytes which have undergone maturation into small (0.6-0.8 mm diameter) oocytes induces germinal vesicle (GV) breakdown. This process has been cytologically studied. Injection of cytoplasm from such small oocytes into large oocytes (1.0-1.2 mm diameter) is followed by the formation of a large spindle bearing many chromosomes at the site of injection.

Induction of maturation (meiosis) in small Xenopus laevis oocytes by injection of maturation promoting factor.

Treatment of small-(stage III) or medium-sized (stage IV) Xenopus laevis oocytes with progesterone, human chorionic gonadotropin, or para-hydroxymercuriphenylsulfonate does not induce maturation. Only the full-grown oocytes (stage VI) undergo maturation when treated with either one of these three substances. In contrast, injection of maturation promoting factor into oocytes of stages III-VI invariably leads to chromosome condensation and germinal vesicle breakdown. No maturation spindle is found in oocytes smaller than 0.9 nm in diameter, and the nuclear sap does not mix with the cytoplasm in the smallest (0.45-0.55 mm in diameter) oocytes. In oocytes of 0.9 mm, maturation is identical to that of full-grown oocytes, except that the maturation spindle does not reach the cortex of the oocyte. Progesterone increases protein synthesis in medium-sized (0.8 mm in diameter) oocytes without inducing meiosis. It has little or no effect on protein synthesis in smaller oocytes.

[Meiosis: role of a histone kinase in the condensation of ovarian oocyte chromosomes of Xenopus laevis and Ambystoma mexicanum]. / Méiose: rôle d'une histone-kinase dans la condensation des chromosomes d'oocytes ovariens de Xenopus laevis et d'Ambystoma mexicanum

By injecting heterologous histone-kinase preparations into ovarian Axolotl oocytes, it has been possible to speed up the progesterone-induced process of chromosome condensation. Moreover, in some instances, this condensation and even complete maturation have been obtained after injection of protein kinase alone, thus in the absence of hormone stimulation. Two different histone kinase preparations have been used: one was prepared from ascites cell chromatin and the other from in vitro ovulated Xenopus oocytes.

Induction of maturation (meiosis) in Xenopus laevis oocytes by three organomercurials.

Three organomercurials, p-hydroxymercuribenzoate, p-hydroxymercuriphenylsulfonate, and mersalyl, induce maturation (meiosis) in a large percentage (20-100 percent) of Xenopus laevis oocytes. Maturation takes place even when the follicle cells which surround the oocytes have been withdrawn. Organomercurial- and progesterone-induced maturations have many features in common: they do not occur when the inducer is injected into the oocytes, they require the presence of Ca++ in the medium, they are inhibited by cycloheximide but not by actinomycin D. In both cases, the maturation producing factor and the pseudomaturation inducing factor are produced. Organomercurial-treated oocytes react normally to activating stimuli; their protein synthesis increases, but uptake of amino acids is strongly inhibited. Progesterone and p-hydroxymercuriphenyl-sulfonate act synergically in inducing maturation. The main difference between the two agents is that p-hydroxymercuriphenylsulfonate must act for several hours, whereas, short contact with progesterone is sufficient to induce maturation.

Characterization of yolk DNA from Xenopus laevis oocytes ovulated in vitro.

High molecular weight DNA was isolated from the yolk platelets of Xenopus laevis oocytes ovulated in vitro. Yolk DNA has the same buoyant density in CsCl gradients as mitochondrial and nuclear DNAs, and, like them, it is double-stranded. Yolk DNA behaves like nuclear DNA, and not like mitochondrial DNA, upon renaturation after denaturation. The molecules are always linear. Cytochemical and biochemical controls preclude the possibility that the yolk DNA might be contaminated by nuclear or mitochondrial DNA. We conclude that the yolk DNA is an endogenous component of the yolk platelets.