In this study we analyzed the relationship between detergent-resistant microdomains and caveolae in Caco-2 cells. Caveolin was not detected on Western blots or Northern blots or by immunoprecipitation in these cells, in contrast to A 431 cells. Triton X-100-resistant membranes from Caco-2 and A 431 cells showed the same morphological aspect by electron microscopy and peaked at the same isopycnic density on sucrose gradients. Detergent-resistant microdomains from Caco-2 cells were enriched in glycosyl phosphatidylinositol (GPI)-anchored proteins, in sucrase-isomaltase, an apical marker, and in most of the proteins found in caveolin-rich membranes such as src-like proteins, fimbrin, ezrin, and Gi alpha. Caveolae-like structures were present in A 431 but absent from Caco-2 cells at the electron microscopic level. Detergent-resistant microdomains from Caco-2 cells resemble caveolin-rich microdomains in their molecular composition but do not seem to derive from morphologically identified caveolae. Our results also indicate that caveolin is not necessary for sorting of GPI-linked proteins to the apical membrane of Caco-2 cells.

Prophase I of meiosis was studied in the human oocyte obtained from 16-to 24-week-old fetuses. Electron microscopy and silver staining showed that, at pachytene, the ribosomal genes belonging to several chromosomes are gathered in the same nucleolar fibrillar center, where they are embedded in an argyrophilic protein. The nucleolus showed spontaneous segregation of its components due to temporary inactivation of the ribosomal genes. The fibrillar center, separated from the other nucleolar components, was penetrated at midpachytene by chromatin fibers containing rDNA emanating from one to three nucleolar bivalents. Thus, the ribosomal genes from 4-12 chromatids are temporarily juxtaposed inside the same structure. Such a structural arrangement is completely different from that observed in the pachytene-stage mouse oocyte, where two independent and active nucleoli, each displaying its own fibrillar center, were formed on the bivalents containing paired ribosomal genes. These different structural patterns' are correlated with the high frequency of nondisjunction in the human oocyte and the relative infrequency of such in the mouse oocyte. The pattern observed in the human oocyte may be a cause of translocations.Chromosomal anomalies due to meiotic nondisjunction are the most frequent cause of human spontaneous abortions (1-4). In contrast, aneuploidy is seldom observed in mouse embryos (5). This difference is explained by the very low incidence (<1.0%) of nondisjunction during the first meiotic division of mouse oocytes (6). Chromosomes containing a nucleolus organizer are involved in about 40% of human lethal trisomies (7). In Down syndrome, the extra chromosome 21 originates, in most cases, as a result of nondisjunction during the first meiotic division of the oocyte (8-10). These data suggest that, at prophase I of human meiosis, a structural arrangement may exist favoring the failure of disjunction of chromosome 21 and other nucleolus-organizer-containing chromosomes. The nucleolar association of several bivalents at pachytene has been described previously (11), but the techniques used gave no information about the part of the nucleolus involved in the association and the possible entanglement of the chromosomal and nucleolar structures. This paper describes a comparative study of the behavior of nucleolus-organizer-containing chromosomes at prophase I of meiosis in the human and the mouse oocyte that was made in an attempt to elucidate the nature of these structural arrangements.MATERIALS AND METHODS Materials. Eight human fetal ovaries taken at 16-24 weeks of pregnancy and eight ovaries from Swiss OF1 mouse embryos taken at the 18th day of pregnancy were used.Silver Staining of the Nucleolar Organizer Region. After isolation and spreading (12), the human germ cells were stained (13) by treatment with 50% silver nitrate solution at 60-700C for 6 min followed by treatment with ammoniacal silver/3% neutralized formalin, pH 5-6. Electron Microscopy. The ovaries were cut into 1-mm3 pieces and fixed by ...

No abstract

Use of the silver-NOR method to study the nucleolar organizers in human oocytes demonstrates that topographic and quantitative variations occur during meiotic prophase. In the oogonia nucleolus the nucleolar organizers are dispersed, whereas beginning at leptotene and throughout the remaining stages of meiotic prophase they occupy a marginal position in the nucleolus. At leptotene, a modal number of seven nucleolar organizers can be observed, whereas this number falls to 2.5 at pachytene and rises to ten at diplotene, thus showing that there is intense rRNA synthesis during the latter stage of meiosis. During pachytene, one end of the bivalents containing the ribosomal cistrons is always associated with the Ag-positive zone of the nucleolus. Observation of pachytene in the electron microscope shows that the secondary constriction region of D and G bivalents is constantly associated with the fibrillar center of the nucleolus. Comparison of these two methods of investigation reveals that the silver-stained regions of the nucleolus correspond to the fibrillar centers. The latter are surrounded by a layer of electron-dense fibrils corresponding to the zone of rDNA transcription. This electron-dense layer is absent during pachytene when the nucleolus displays spontaneous segregation of its components; this absence is related to temporary arrest of rDNA transcription. The affinity of the fibrillar centers for silver-NOR staining confirms that these structures contain ribosomal cistrons. During the diplotene stage, numerous micronucleoli are formed outside the nucleolar organizers of D and G chromosomes. Most of these micronucleoli present an Ag-positive granule on one of their margins, thus indicating that they contain an actively transcribed sequence of rDNA. This observation confirms the existence of amplification of ribosomal genes in the human oocyte.

By means of immunocytochemistry with a battery of specific antibodies, we describe the expression of the collagen IV chain produced by the gene DCg1 during the two phases of Drosophila neurogenesis. DgC1 was not expressed in neuronal tissues as shown by in situ hybridization, but the onset of its expression in cells of mesodermal origin was concomitant with the appearance of collagen IV on early axon pathways and peripheral nerve roots in the embryonic developing CNS. A similar situation was found during imaginal CNS development at metamorphosis, when collagen IV immunoreactivity was detected on centrifugal and centripetal nerve pathways, and specially on retinula axons that develop from the eye imaginal disc towards the lamina anlage in the brain optic lobe. Our results strongly suggest that collagen IV could be involved, together with other informative molecules of basement membranes, in a dynamic process of cell-matrix interactions during the establishment of initial axon pathways and neurite outgrowth in vivo.