Jean-Claude Lacroix

Cytogenetic and genetic evidence of male sexual inversion by heat treatment in the newt Pleurodeles poireti

Larvae of Pleurodeles poireti were maintained during their development at a high temperature (31° C). In several species of amphibians, such a treatment is known to change the sex ratio through the inversion of genotypic females into phenotypic males. Pleurodeles poireti is an exception. It is the first reported amphibian in which heat induces an inversion of genotypic males into functional phenotypic females. The sexual genotype of standard and experimental phenotypic females was determined through heterochromosomes in lampbrush stage. In the present study, we have utilised another technique for identification of sexual genotype, applicable to both phenotypic males and females. It is based on the differential expression of a sexlinked gene, the peptidase 1.

A novel approach to cytotaxonomic and cytogenetic studies in the genus Triturus using monoclonal antibodies to lampbrush chromosomes antigens

Monoclonal antibodies against germinal vesicle antigens from Pleurodeles oocytes crossreact with lampbrush chromosomes of various Triturus species: C36/6, A33/22 and B71/22 bind to most lateral loops, B24/3 labels the spheres, while A1/5 and B81 give a distribution of fluorescent loops which is highly reproducible and species specific. — The antigens involved were characterized by immunoblotting of electrophoretic gels of germinal vesicle proteins and the molecular weights of those that bound to monoclonal antibodies C36/6, A33/22, B24/3 and C3/1 were determined. — The possible relationship between sites immunostained by some monoclonal antibodies and given DNA sequences distributed along the chromosomes is discussed. A new approach to cytotaxonomic and cytogenetic studies through the use of monoclonal antibodies on lampbrush chromosomes is offered, which can give new insight into the molecular mechanisms of speciation and karyological evolution in European newt species.

Behavior of a Deionized Liquid Crystal Subjected to Unipolar Injection in Nematic and Isotropic Phase

Electrohydrodynamic phenomena in insulating MBBA subjected to unipolar injection of ionic carriers are described. Near the transition temperature, the transient currents following a voltage step were similar in the nematic and isotropic phases, over a wide range of voltages and electrode separations. High‐speed cinematography, Schlieren techniques, and the Kerr effect were resorted to in this investigation. Liquid motion was observed having a time lag and exhibited patterns closely similar to those previously observed in isotropic polar liquids. The velocities of hydrodynamic disturbances and of space‐charge fronts varied over a broad range, depending on experimental conditions.

Spontaneous sex reversal in Pleurodeles waltl (urodele amphibia): analysis of its inheritance

Up to now, no case of spontaneous sex reversal has been described in the urodele amphibian Pleurodeles waltl reared at ambient temperature. However in offspring reared under laboratory conditions, males, females but also intersex individuals were obtained. The males, some females and the intersexes had the ZZ male genotype identified through a test performed with a sex-linked enzyme. The other females had the ZW female genotype. Using the animals of this particular strain, an offspring analysis was made on crosses between, respectively, ZZ males and ZW females, ZZ males and ZZ females, and ZZ intersexes and ZZ males or females. All these crosses gave ZZ males, ZZ females and ZZ intersexes. The spontaneous complete or partial sex reversal is inheritable, but the genetic mechanism of this inheritance has not yet been elucidated.

Localization of antigens PwA33 and La on lampbrush chromosomes and on nucleoplasmic structures in the oocyte of the urodele Pleurodeles waltl: Light and electron microscopic immunocytochemical studies

Monoclonal antibodies A33/22 and La11G7 have been used to study the distribution of the corre-sponding antigens, PwA33 and La, on the lampbrush chromosome loops and nucleoplasmic structures of P. waltl oocytes, using immunofluorescence, confocal laser scanning microscopy and immunogold labeling. The results obtained with these antibodies have been compared with those obtained with the Sm-antigen-specific monoclonal antibody Y12. All these monoclonal antibodies (mAbs) labeled the matrices of the majority of normal loops along their whole length. Nucleoplasmic RNP granules showed a strong staining with the mAbs La11G7 and Y12 throughout their mass, but with the mAb A33/22, they showed only a weak peripheral labeling in the form of patches on their surface. This patchy labeling was confirmed by confocal laser scanning microscopy. Electron microscopy revealed that this patchy labeling might be due to a hitherto undescribed type of submicroscopic granular structure, around 100 nm in either dimension, formed by 10-nm particles. Such granules were observed either attached to the RNP granules or free in the nucleoplasm, but rarely in relation with the normal loop matrices. These 100-nm granules may have a role in the movement of proteins and snRNPs inside the oocyte nuclei for storage, recycling, and/or degradation. Our results also suggest that all the microscopically visible free RNP granules of the nucleoplasm of P. waltl oocytes correspond to B snurposomes. The granules forming the B (globular) loops showed a labeling pattern similar to that of B snurposomes; their possible relationship is discussed.

B24 protein stored in lampbrush spheres is involved in early cleavage in urodele amphibians

A cDNA encoding a protein (B24) belonging to the Mcm/P1 family was isolated from the newt Triturus carnifex. In eukaryotes, the members of the Mcm/P1 family are essential factors in the DNA replication process. B24 protein (TcMcm3) is present in salamandrid ovarian oocytes and early embryos; its role was tested by injecting specific anti-B24 monoclonal antibodies into the cytoplasm of one blastomere of two-cell stage embryos. The injected blastomere encountered cleavage arrest either soon after the injection or following one or two divisions; later, it degenerated. Instead, the uninjected blastomere went on developing and organizing a hemi-embryo, which does not grow beyond the tailbud stage. These results are consistent with the hypothesis that the B24 protein is involved in DNA replication at cleavage. The B24 protein studied here appears to play a specific role in early development; other variants of the Mcm3 group seem to be employed by different adult tissues.

Correlation between isolated lampbrush chromosomes and nuclear structures of Pleurodeles waltl oocytes: An electron microscopic study

The organization of the lampbrush chromosomes of Pleurodeles waltl was studied by fixation and embedding of oocytes in toto and correlated with that observed in end-embedded preparations of isolated chromosomes. Particular attention was focused on marker loops, like the granular and globular loops, and atypical structures known as spheres (S) and M. In both types of preparations, the majority of the loops, the so-called normal loops, and the granular loops appeared to share a common basic organization, with ribonucleoprotein (RNP) fibrils appearing as strings of 30 nm particles, as described by earlier authors. Some new types of loop organization were observed: (i) P loops with 45 nm RNP particles; (ii) dense granular loops; (iii) loops with a cylindrical organization. RNP fibrils formed by 60 nm particles were found to occur in association with the globular loops. EDTA staining suggested the presence of large amounts of RNP in the sphere but very little in M. Three morphologically different types of RNP granules could be observed free in the nucleoplasm.

Light and electron microscopic immunolocalization of two nuclear antigens in the liver of Pleurodeles waltl using monoclonal antibodies

The distribution of 2 nuclear antigens in the interphase nuclei of liver of Pleurodeles waltl was determined with the help of monoclonal antibodies, using immunofluorescence for light microscopy and indirect immunoperoxidase and immunogold labeling procedures for electron microscopic localization. The antibodies C36/1 and A33/22 label antigens with relative molecular masses of 270 kDa and 80 kDa, and isoelectric points of 7.0 and 6.4, respectively. The liver of urodels is characterized by the presence of a peripheral layer of hematopoietic cells around the parenchymatous tissue formed by typical hepatocytes. The antibody C36/1 labels the nuclei of both types of cells, whereas the antibody A33/22 labels the nuclei of hepatocytes but not those of the peripheral hematopoietic cells. With both these antibodies, labeling, whenever observed, is restricted to fibrillar structures in the interchromatin space, i.e., to peri‐ and inter‐chromatin fibrils; condensed chromatin, nucleoli, and nuclear envelope are not labeled.

A new function for the spheres of newt lampbrush chromosomes

Abstract Nuclei of amphibian oocytes contain spheres, either attached at specific loci of lampbrush chromosomes or free in the nucleo‐plasm, the structure and function of which have been investigated for long time. These spheres were initially shown to contain components involved in pre‐mRNA splicing, pre‐rRNA processing and histone pre‐mRNA 3’ end formation. Later, the spheres were labelled by B24/3 monoclonal antibody, which recognizes a maternal protein of 104 kDa. Recently, a cDNA clone encoding protein B24 has been isolated in Triturus carnifex and analysis of the nu‐cleotide sequence has shown that B24 protein belongs to the Mcm/P1 family, which is thought to play an important and conserved role in initiation of DNA replication. To test the function of B24 protein microinjections of B24/3 mAb were made into one blastomere of two and four‐cell stage embryos. The injected cell degenerated and only hemi‐embryos or underdeveloped embryos were obtained, because B24 protein plays an important role in cell...