Jean-Claude Beetschen

Red blood cells and hemoglobin concentration in normal diploid and several types of polyploid salamanders

Abstract 1. 1. Different blood parameters were examined in both diploid and polyploid adult Pleurodeles newts. 2. 2. In 2 n, 3 n and 4 n, the hemoglobin content and hematocrit values were very close. 3. 3. The red blood cell counts decrease as the degree of polyploidy rises. 4. 4. Autotetraploid and pentaploid animals do not regulate their hemoglobin values and are slightly anaemic.

A three-step scheme for gray crescent formation in the rotated axolotl oocyte☆

It has been shown that various inhibitors of protein synthesis can elicit the precocious appearance of a gray crescent (GC) in in vitro maturing, nonactivated Ambystoma mexicanum oocytes. However, evidence has now been obtained that these treatments fail to induce GC formation when the oocytes are enucleated before initiation of maturation. The ability to form a GC is reestablished in enucleated oocytes by the injection of nucleoplasm from a normal oocyte, either before or after the injection of the inhibitor. In the latter case, the GC appears very rapidly, even though protein synthesis is at about 1/10th that of the control enucleated oocyte, after treatment with diphtheria toxin (final concentration 10(-8) M) as an inhibitor. One or several nuclear factors, in conjunction with inhibition of protein synthesis, are therefore essential for early symmetrization. The corrective nuclear factor is already present in the germinal vesicle of young oocytes, at the very beginning of vitellogenesis. It is not species specific, since enucleated axolotl oocytes can be symmetrized with Pleurodeles or even Xenopus oocyte nucleoplasm. Moreover, it has been shown that the nuclear-cytoplasmic interaction is possible only when cytoplasmic maturation has been proceeding for at least 10 hr after exposure to progesterone (at 18 degrees C). A three-step process as a prerequisite of GC formation in the oocyte is proposed: Cytoplasmic maturation must proceed till a reactive state is attained, allowing interactions with nuclear factors; Nuclear factor(s) interact(s) with matured cytoplasm; Inhibition of protein synthesis triggers GC formation. Sequence of steps 2 and 3 can be experimentally inverted but must always be preceded by step 1. Since a sharp reduction in amino acid incorporation has also been found in normally fertilized eggs just prior to GC formation, it is suggested that the scheme described above could be also applicable to normal symmetrization in this model system.

Action du Facteur de croissance nerveuse (Nerve Growth Factor) sur la différenciation de cellules embryonnaires d'amphibien

The in vitro effects of NGF extract on determinate and indeterminate cultured cells of Pleurodeles waltlii ((Amphibian, Urodela) are studied. Cytological changes at nuclear and cytoplasmic levels are observed. The formation of neurons induced by NGF in undeterminated cell cultures (young gastrulas presumptive ventral epiblast), plus the presence of a particular cell type in determinated cell cultures (young neurulas neuroblast and chrodomesoblast) are described. We witnessed for this factor a neuron growth stimulating effect. The cytological alterations and the effects on morphological differentiation are irreversible. A few hypotheses for NGF mechanism are proposed.

A maternal-effect mutation disturbs extracellular matrix organization in the early Pleurodeles waltl embryo

SummaryIn the early development of the urodele amphibian Pleurodeles waltl, a fibronectin-containing extracellular matrix underlies the inner face of the blastocoel roof. When gastrulation occurs, the fibronectin fibrils provide a suitable substrate for mesodermal-cell migration. Delay in morphogenetic movements of gastrulation has been described in embryos from mutant females (ac/ac) of Pleurodeles waltl. Studies of abnormal mutant gastrulae with fluorescent lectins and immunostaining for fibronectin reveal that they lack a normal matrix. The fibronectin-containing extracellular material always gives rise to a granular pattern without fibronectin-fibril formation. Fibronectin and α5β1 syntheses occur normally in maternal-effect embryos. In vitro, mesodermal cells from early mutant gastrulae adhere and migrate on fibronectin-conditioned substrata.

Lithium induces dorsal-type migration of mesodermal cells in the entire marginal zone of urodele amphibian embryos

SummaryThe effect of lithium (Li+) on gastrulation movements was investigated during the development of the urodele amphibianPleurodeles waltl. Attention was focused on mesodermal cell migration. Under conditions of Li+ treatment providing a maximal enhancement of dorsoanterior structures, it was found that the dorsoventral polarity of gastrulation was abolished. In particular, vital staining and scanning electron microscopy observations on embryo fractures showed that mesodermal cells migrated radially after Li+ treatment, which led to the formation of rounded embryos. Epiboly movements thus were accelerated. Nevertheless, contrasting with the precocious disappearance of the early-formed yolk plug, archenteron invagination was constantly retarded and commenced with a delay of several hours as compared to control gastrulae. Cell-lineage analysis of the progenies from ventral or dorsal equatorial blastomeres of 32-cell-stage embryos provided evidence that both dorsal and ventral mesoderm contributed to notochordal tissue after Li+ treatment. Dorsalization of the entire marginal zone was confirmed by the ability of the entire mesoderm rudiment to behave as a dorsal organiser after Li+ treatment. Comparison of the migratory behaviour of isolated animal hemispheres from Li+-treated or control embryos cultured on fibronectin-coated substrate indicated that all marginal cells acquired the autonomous capacity for migration of dorsal marginal cells under the action of lithium.

Inhibition of protein synthesis elicits early grey crescent formation in the axolotl oocyte

SummaryIn the axolotl (Ambystoma mexicanum Shaw), it was recently shown that cycloheximide (CH) could induce early grey crescent formation (EGC) in non-activated oocytes, maturing in vitro (Grinfeld and Beetschen 1982). Since it was not proved that EGC was a consequence of protein synthesis inhibition rather than a side-effect of the drug, experiments were performed using microinjections of a quite different inhibitor, diphtheria toxin (DT). This toxin also appeared to elicit EGC. Incorporation of (3H) leucine into oocyte proteins in the presence of increasing DT concentrations (10−11 to 10−6 M) was then investigated. The frequency of EGC closely parallels the level of protein synthesis inhibition, which is higher in symmetrized oocytes. The lowest CH concentration which can still elicit EGC also exerts a fairly strong inhibition of (3H) leucine incorporation into proteins. It is concluded that protein synthesis inhibition in the late maturing oocyte actually creates specific conditions which allow cytoplasmic rearrangements to occur, leading to grey crescent formation. These results support the interpretation that (a) proteinic inhibitor (s) of symmetrization could be synthesized in the normal maturing oocyte.

Early grey crescent formation experimentally induced by cycloheximide in the axolotl oocyte

SummaryThe effects of cycloheximide (CH) on grey crescent formation in artificially maturedAmbystoma mexicanum oocytes were determined. CH induced grey crescent formation after a few hours, especially after a 45° to 90° rotation from the vertical animal-vegetal axis. With low concentrations of CH (about 0.5 ng/oocyte), meiosis was still able to proceed normally to the stable second metaphase stage, but higher concentrations blocked it after 1st polar body extrusion and an interphasic nucleus appeared. Such effects were compared to those of inactone, an analogue of cycloheximide, which as a pure substance does not inhibit protein synthesis, but still contained a small amount of CH in the available samples. It is concluded that grey crescent formation can occur in non-activated oocytes. The effects of cycloheximide might be due to partial inhibition of protein synthesis and the presence of a proteinic inhibitor of the symmetry reaction in the normal oocyte is suggested.

Effets Inhibiteurs de Protéines Chromatiniennes non Histoniques Homospécifiques, sur la Morphogenèse d'Embryons d'Amphibiens Urodèles

Non-histone chromosomal proteins (NHCP) were injected into the blastocoel of advanced blastulae fromPleurodeles waltlii which were previously punctured. NHCP extracted from the liver of the same amphibian species bring very strongly inhibitory effects on morphogenesis: gastrulation is prevented in 75% of cases, but the embryos can survive for 8 to 10 days without showing any necrotic cells, and cell divisions still occur sporadically. Intercellular adhesivity is strongly impaired in such embryos, in connection with inhibition of morphogenetic movements. Gastrulation is delayed and abnormal in other embryos, and neurulation is severely disturbed, the neural tube being unclosed or much reduced in size and the notochord and somites poorly differentiated. In contrast, NHCP extracted from other animal species (rat and chicken liver, rat ascites) have practically no effects onPleurodeles embryos. Species-specific inhibitory effects are thus demonstrated and compared with those which were previously studied on cultures of differentiating embryonic cells fromPleurodeles waltlii andAmbystoma mexicanum. The possible levels at which NHCP act are discussed, the changes in intercellular adhesivity being noted as more conspicuous in the present experiments.

[Chromosome studies of Tylototriton verrucosus Anderson and of viable hybrids from Pleurodeles waltlii (female) x Tylototriton verrucosus (male) (urodele amphibians, Salamandridae)].

The karyotypes of T. verrucosus and of the viable intergeneric hybrid between P. waltlii ♀xT. verrucosus ♂ have been made up from different kinds of colchicinic metaphases (somatic larval cells and spermatogonia). T. verrucosus has no pair of telocentric chromosomes corresponding to the description previously made on another species, T. andersoni. — In T. verrucosus, a particular chromosome generally does not exhibit much morphological variability according to its origin, from a spermatogonial or a somatic cell. The chromosomes of T. verrucosus (2n=24) can be classified into three groups, each group being made of four pairs, like those of P. waltlii. The mean relative lengths and arm-ratios are nearly identical in both species for most of the chromosomes, except Nos. (2), (6) and (12): in T. verrucosus, the arm-ratio is higher than in P. waltlii for somatic chromosomes (2) and (6), and for gonial chromosomes (6), but is lower for somatic chromosomes (12). A few differences between the two species can be seen in the position of secondary constrictions which appear on somatic chromosomes after a cold treatment: in T. verrucosus, one constriction on each arm of chromosome (3), no constriction on the long arm of No. (6), one constriction on the short arm in most of the chromosomes (7), no constriction on No. (12). — In hybrid metaphases, chromosome (6) from Tylototriton is the only one which can be identified with certainty without a cold treatment. When secondary constrictions are present, it is possible to distinguish between the two components of pairs (3), (12) and occasionally (7), the two chromosomes (6) being always characteristic. — From triploid hybrid metaphases, it seems that the absolute length of individual chromosomes of T. verrucosus could be greater than that of homologous maternal chromosomes. — Problems of compatibility between the genetic characters of these related but geographically separated genera are shortly discussed.

Differential Protein Distribution Related to Dorsoventral Polarity in Pleurodeles waltl Cleaving Egg

The molecular basis for the initial specification of dorsoventral polarity in the Amphibian egg prior to the mid‐blastula transition still remains an open question. Regional differences in the protein pattern of Pleurodeles egg were investigated during early cleavage (8‐ and 512‐cell stages, prior to the mid‐blastula transition). Animal‐dorsal, animal‐ventral, vegetal‐dorsal and vegetal‐ventral quarters were separated and proteins were analyzed by 2D‐electrophoresis. The comparison of acidic protein patterns from dorsal and ventral quarters revealed differences between vegetal cells but no difference was detected between animal cells. One protein (p11, 30 kDa) was characterized in the dorsal side as early as the 8‐cell st. and two dorsal spots were detected at the 512‐cell st. (p11 and p5, 65 kDa). Similarly one protein (p7b, 46 kDa) appears to be ventral‐specific from the 8‐cell st. The p11 spot was shown to appear in ventral cells as a consequence of a dorsalizing LiCl‐treatment at the 32‐cell stage. Conversely, p11 disappeared from dorsal cells and p5 did not appear at 512‐cell stage after UV‐irradiation of the uncleaved egg, which results in the expression of the ventral‐specific protein p7b in the dorsal part of the egg. Therefore differential protein expression is already present at very early cleavage stages. Its significance needs further investigation.