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.

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.

Presence of rRNA in the heavy bodies of sea urchin eggs. An in situ hybridization study with the electron microscope.

Experimental conditions have been found, in which the presence of rRNA can be demonstrated by in situ hybridization at the electron microscope level in the heavy bodies of sea urchin eggs. The specificity of hybridization has been controlled by ribonuclease digestion and by competition experiments with unlabelled rRNA.

Studies on differentiation without cleavage in Chaetopterus. Requirement for a definite number of DNA replication cycles shown by aphidicolin pulses.

Aphidicolin, a powerful inhibitor of nuclear DNA replication, has been used to establish the level of polyploidisation required for the realization of the late morphogenetic events (segregation, pseudogastrulation and ciliation) of differentiation without cleavage in Chaetopterus-activated eggs. A parallel has been drawn between cytophotometric estimations of the DNA content and cytoplasmic differentiation in eggs treated with aphidicolin under different experimental conditions: either pulses with aphidicolin were followed by culture in sea water or the eggs were treated with aphidicolin after development had taken place in sea water for various lengths of time. The results suggest that a 'quantal' monasterial cycle might take place 3 h after activation, corresponding presumably to the fourth or fifth replication cycle. Moreover, early DNA replication seems to be more important for morphogenesis than late DNA replication.

Studies on differentiation without cleavage in Chaetopterus: effects of inhibition of DNA synthesis with aphidicolin.

The effects of aphidicolin - a powerful inhibitor of DNA polymerase alpha and of DNA replication - on normal development and on differentiation without cleavage of Chaetopterus eggs have been studied with cytological, cytochemical, and biochemical methods. The experiments show that the initial period of pseudocleavage can take place in the absence of nuclear DNA synthesis, but further development (segregation, hatching, ciliation) requires DNA synthesis. However ciliated unicellular larvae can be obtained under conditions where the DNA content of the embryos in only 40% of the controls. In fertilized eggs, aphidicolin immediately stops cleavage. The significance of these results is discussed.

Studies on differentiation without cleavage in Chaetopterus variopedatus: requirements for nucleic acid and protein synthesis.

Activation of unfertilized Chaetopterus eggs by treatment with an excess of KCl may lead to the production of unicellular ciliated larvae (Lillie's differentiation without cleavage). The effects of a number of inhibitors of protein (puromycin, cycloheximide, emetin), RNA (actinomycin D), and DNA (hydroxyurea) synthesis of differentiation without cleavage and on normal development have been studied in Chaetopterus. Incorporation of radioactive leucine, uridine, and thymine has been followed by biochemical methods and by autoradiography. The DNA content of the large polyploid nucleus has been estimated by cytophotometry. The initial pseudocleavage period of differentiation without cleavage is characterized by a burst in DNA and protein synthesis; the inhibitors have little or no effect on this burst and on pseudocleavage itself. Protein and DNA synthesis levels off during the following phase (segregation), but the inhibitors become more effective. RNA synthesis is almost linear for 20 h. These results are compared with those obtained on eggs from other species.

The hormonal induction of maturation in amphibian oocytes.

The maturation of the amphibian oocyte has been analyzed. Progesterone as well as organomercurials, lanthanum chloride and propranolol rapidly induce maturation. These chemicals are active only is applied on the cell surface. The mechanism seems to be an induction of the migration of Ca2+ from the cell membrane to the cytoplasm. K + may also play a role. Progesterone induced maturation involves synthesis of histone and histone kinase as well as several biologically active but chemically unidentified factors. cAMP does not seem to be directly involved, whereas protein phosphorylation is so.

[Effect of valinomycin on mitotic activity and ciliary movement during embryonic development]. / Effets de la valinomycine sur l'activité mitotique et le mouvement ciliaire pendant le développement embryonnaire.

The K+ ionophore valinomycin very quickly arrests cleavage in sea urchin and mouse eggs at concentrations ranging between 10 and 3 micron. Development of Axolotl and Xenopus eggs is not arrested before the blastula or gastrula stage. The motility of sea urchin sperm, blastulae and gastrulae is suppressed, within a few minutes, by 1-9 micron valinomycin.

Cytochemical study of human lymphocytes stimulated by PHA in function of donor age.

The 3H-AM binding reflects the structural changes involved in the cellular differentiation. This parameter was studied during blastic transformation of human lymphocytes, in relation to the age of the donor. Although they are individual variations, the 3H-AM binding is higher in the young group than in the aged subjects, as well as the blast transformation score. These results indicate that the weak lymphocyte response to the mitogen lectine (PHA) stimulation could be related to some age-induced structural alterations of the chromatin, resulting in an irreversible blockage in G1, at least in some of the T lymphocytes.

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.

[The effects of diverse agents (Ca++ and K++ ionophores, organomercurials, dithiols, colchicine, and cytochalasin B) on the maturation and differentiation without cleavage in Chaetopterus eggs]. / Effets de divers agents (ionophores du Ca++ et du K+, organomercuriels, dithiols, colchicine et cytochalasine B) sur la différenciation sans clivage chez l'oeuf de chétoptère.

Induction of maturation in Chaetopterus oocytes requires the presence of Ca++ ions in the medium, but differentiation without cleavage can proceed in the absence of this cation. The Ca++ ionophore A 23187 induces both maturation and the cortical reaction provided that Ca++ ions are present in the medium differentiation without cleavage may follow. Valinomycin slowly induces germinal vesicle breakdown, which is followed by a sharp segregation between hyaloplasm and yolk. PHMPS, but not DTT, induces maturation. Differentiation without cleavage is more sensitive to colchicin than to cytochalasin B.