Alain Lepoint

Behaviour of nucleolus during mitosis. A comparative ultrastructural study of various cancerous cell lines using the Ag-NOR staining procedure.

The aim of the present work was to study the distribution and the behaviour of the silver-staining nucleolar organizer region (Ag-NOR) proteins at the ultrastructural level during interphase and mitosis in five human and murine cancerous cell lines each characterized by a typical nucleolar morphology. During interphase the Ag-NOR proteins are restricted to the fibrillar centres (F.C.) and/or to the dense fibrillar component (D.F.C.). During prophase the silver-staining components come into close contact with some chromosomes and are arranged with a typical polarity: chromosome, F.C. and D.F.C. Then F.C. and D.F.C. together form roundish silver-stained structures and integrate in part within indentations at the periphery of the metaphase chromosomes. During anaphase and telophase large and small spherical silver-staining structures may be seen. They correspond respectively to the metaphase NORs and to numerous structures which appear de novo within ribonucleoprotein (RNP) material localized between the chromosomes. During late telophase the number of the small silver-staining structures decreases whereas the size of the larger ones increases. Then the interphase nucleoli recover their typical shape. These results suggest that when rRNA synthesis is impaired during mitosis the inactive NORs assume a structure and a localization which are not typical of the cell line. In contrast the F.C. and D.F.C. are probably two aspects of the NORs whose typical distribution, relative to the other nucleolar components, gives the interphasic nucleolus its characteristic morphology.

Ultrastructural cytochemistry of the nucleolus in rat oocytes at the end of the folliculogenesis

SummaryVarious ultrastructural changes occur during follicular growth in the rat oocyte nucleolus. The nucleolus, which has a reticulated fibrillogranular structure at the primordial and primary follicle stages, becomes entirely compact and is made up of a conspicuous and homogeneous mass at the antral follicle stage. In order to define the nature and the functions of this homogeneous mass, cytochemical methods allowing detection of nucleic acids, proteins and lipids were performed at the light microscopic and ultrastructural levels. The results obtained suggest that this nucleolar mass is probably composed of acid proteins which are not silver stained. This proteinaceous mass could be a special kind of nucleolar secretion providing material for meiotic resumption in the oocyte. Cytochemical researches now in progress should supply new information concerning the exact nature and the role of the nucleolar compact mass, which is the essential nucleolar component at the antral follicle stage and which really plays a role in the nucleolus in the first stages of embryogenesis.

Relations between nucleoli and nucleolus-organizing regions during the cell cycle

A clear relationship between nucleolus and some chromosomal regions was first established by Heitz (1931). This author demonstrated the involvement of these chromosomal regions in the telophase reconstitution of the nucleoli. McClintock (1934) called these regions “nucleolus organizing regions” (NORs), a term indicating that the nucleoli originate in these regions.

Effects of ethidium bromide on chick fibroblasts and mouse Ehrlich tumor cells cultivated in vitro. Cytological and cytochemical observations.

Summary Normal chick embryo fibroblasts and Ehrlich ascites tumor cells have been cultivated in vitro in the presence of ethidium bromide (E. B.). Cell growth is strongly inhibited and some cells die. However, the modal cellular volume does not change. The mitochondria are swollen, fragmented and sometimes spherical; many cristae are destroyed and large granular inclusions are present in the matrix. The nucleoli become smaller, denser and the nucleolar constituents are segregated. Some nuclei contain large chromatin blocks. E. B. can be detected in the cell by fluorescence microscopy: it is located in the cytoplasm, in the nucleoli, in the chromatin and inside mitochondria, especially in the intramitochondrial granules. Ethidium bromide can inhibit DNA synthesis and block the cells during DNA synthesis; sometimes, the block happens in G2 (postsynthesis as to DNA). Later on, RNA and protein synthesis can also be inhibited. All these effects are nearly identical in the normal fibroblasts and in the tumor cells.

Number of nucleoli in ehrlich tumor cells during interphase

SummaryFractions of mouse Ehrlich ascites tumor cell populations with a high percentage of cell either in early or in late interphase were separated by centrifugation on ficoll gradients. Nucleoli were studied by light or electron microscopy in these cell subpopulations. It was shown that, in these cells, the number of nucleoli per nucleus does not vary significantly during interphase. This result is discussed and an analysis of the relationships between the number and the volume of the nucleoli in these cells is presented.

Fractionation and characterization of the blast cell population of the mouse thymus by physical cell separation methods and ultrastructural analysis

Abstract Sedimentation at unit gravity and discontinous density gradients were used either separately or sequentially in an attempt to isolate cell preparations that are significantly enriched in one of the several subtypes of thymocytes present in the C57BL mouse thymus. Electron microscopy analysis of the cellular content of fractions obtained by such methods demonstrates that fractionation by 1g sedimentation allows the isolation of either pure preparations of small cortical lymphocytes or of fractions that are significantly enriched in blast cells, whereas density gradient centrifugation yields to the selection of the low to medium density lymphoblasts. A sequential separation procedure using both sedimentation at unit gravity and density gradient centrifugation was found to be the method of choice to isolate pure preparations of medullary lymphoid cells or to obtain fractions devoid of small lymphocytes and highly enriched in blast cells of the X subtype. These methods may thus serve as a basis for the selective isolation of putative target cells which are believed to be present in the thymus and to play a role in the pathogenesis of thymic lymphomas in C57BL mice.