Ultrastructural similarity in landmark loops of amphibian lampbrush chromosomes

Simultaneous transmission and scanning electron microscopy studies were performed on lampbrush chromosomes of Notophthalmus viridescens and Xenopus laevis. The organization of their normal and landmark loop ribonucleoprotein (RNP) matrices was compared to that of Pleurodeles waltl lampbrush loops, previously described. Ultrastructural observations clearly showed that in the three species, the RNP matrix of normal and landmark loops displayed a common basic structure: an RNP fibril packed into tightly juxtaposed RNP particles of remarkably uniform size, ie 30 nm. Furthermore, analysis of the spatial arrangement of these constitutive RNP fibrils allowed us to establish ultrastructural similarities between the different types of loop matrices of the three species studied. Thus, granular loops with the same organization were found to be present in the three species, whereas Pleurodeles was the only one to exhibit, in its lampbrush chromosomes, the typical globular matrices previously described. “Sequential labelling loops” of Notophthelmus were shown to be similar of both “convoluted dense loops” of Xenopus and “dense loops” of Pleurodeles.     

Effects of in vivo heat treatment on lampbrush chromosome structure in amphibian oocytes

When Pleurodeles (Amphibian, Urodele) females were subjected to high temperatures (32-35 degrees C) for varying periods of time (45 min to 7 days), lampbrush chromosome structure underwent striking modifications. These changes included a numerical reduction in normal loops and progressive disorganization of RNP matrices of various loops. The degree of such disorganization was a function of the intensity and duration of the stress. These modifications were completely reversible when females or oocytes were returned to a normal breeding temperature (20 degrees C). Results are discussed in comparison with previous studies on morphological changes induced by heat shock in lampbrush chromosomes carried out in vitro.     

Morphological observations on cultured lampbrush-stage Rana pipiens oocytes.

Early lampbrush-stage oocytes are characterized by small lampbrush chromosome loops, a small amount of ribonucleoprotein (RNP) matrix on the loops, small nucleoli, few RNP particles in the nucleoplasm, and a smooth germinal vesicle contour. In vitro culture of these oocytes in serum-free culture medium for 24 hr at 18°C promotes a number of morphological changes in the oocytes: The lampbrush loops increase in diameter and acquire extensive RNP matrix, the nucleoli increase in size and complexity, the nucleoplasm accumulates numerous polymorphic RNP particles, and the germinal vesicle envelope acquires a sacculated contour. These characteristics are typical of the in vivo maximum lampbrush stage, and their appearance is due to an apparent in vitro acceleration of the lampbrush phase. Two possible interpretations of these observations are discussed.     

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.     

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.     

Heterochromatin regions of the chromosomes of the hen and Japanese quail in mitosis and at the lampbrush stage

The mitotic and lampbrush chromosomes of the domestic fowl and Japanese quail were analysed by fluorochrome staining technique. The lampbrush chromosomes of both the subjects displayed a typical "loop-chromomere" structure. Three distinct kinds of loops were distinguished in Gallus g. domesticus--normal, telomeric bows, and lumps. The former are distributed along the whole chromosome length. The latter and the bows were observed in subtelomeric and telomeric regions. By DNA/RNA specific acridine orange staining it was shown that each loop (especially, "lumpy" loops) contained a rich RNP matrix. A comparative analysis of the chromomycin A3/distamycin A banding pattern of mitotic and lampbrush chromosomes shows that the telomeric "bows" and "lumps" are special loops developed in telomeric heterochromatic bands. In Coturnix c. japonica, the CMA/DA-positive bands were not observed in telomeres of mitotic macrochromosomes, except a smallest band in the 2p-arm telomere. The absence of telomeric heterochromatic bands which can be visualized in the quail mitotic chromosomes coincides with the absence of "bow"-like loops. Only small lump-like structures were seen in some telomeres of macroautosomes. The biological significance of loop formation and RNA synthesis in heterochromatic band loops in growing oocytes is briefly discussed.