Mammalian cells specifically internalize some molecular species through receptor-mediated endocytosis (RME). We have used four different experimental protocols to investigate whether ouabain enters cardiac cells of guinea pig atrium through this pathway. First, by electron microscope morphometry we found that ouabain increased endocytic vesicles in atrial cells. Second, by scintillation counting we found that [3H]ouabain uptake by the tissue is decreased by three treatments that decrease RME, i.e., NH4Cl, trifluoperazine, and 16 mM [K+]0. Third, by radioautography at the electron microscope level, we checked that in preceding experiments [3H]ouabain was washed out of plasma membrane after 60-min rinse and interiorized into the cardiac cells. Fourth, isometric tension recordings showed that the positive inotropic effect of ouabain was diminished in the presence of inhibitors, whereas that of a hydrophobic analogue, ouabagenin, was not affected. These results suggest that ouabain enters cardiac cells through RME and also that an intracellular site may, at least in part, be responsible for its inotropic effect.

The localization of estradiol receptor (ER) in various tissues and their distribution in sub-cellular compartments were studied by means of immunogold-electron microscopic methods using a site-directed polyclonal antibody developed against a peptide from the DNA binding site of ER. This method was used to determine the presence and localization of ER in tissues and cells of male and female reproductive and non-reproductive organs. In the female reproductive tract, endometrial cells and the cells of the corpus luteum were found to contain ER. In non-reproductive organs of both sexes the following cell types showed significant labeling: hepatocytes, epithelial duodenal cells, striated muscle fibers, cells of the proximal convoluted tubules of the kidney, lymphocytes, neurons, and adipose cells. Alveolar epithelial cells were studied only in female specimens and were labeled by the anti-ER. Prostatic and epididymal epithelial cells were found to be labeled in the male reproductive organs. In all these cells a higher density of label was found in the nucleus, especially in the space between the clumps of compact chromatin, as was previously found in epithelial endometrial cells. These results suggest that estradiol exerts its effects through a common nuclear mechanism in cells of male and female reproductive and non-reproductive organs.

The distribution of DNA and RNA in the synaptonemal complex and related structures, was studied using high resolution cytochemical methods and in situ hybridization, in guinea pig and rat testis. Serial sectioning demonstrates that frequently the formation of the synaptonemal complex (SC) occurs without a previous development of isolated chromosomal axes. The lateral elements of the forming SC are in continuity with pairs of DNA-containing thin filaments. These chromatin filaments fold in numerous short loops just before incorporating to the lateral elements. Some of these loops are included in the ribbon-like structure of the lateral elements of the mature SC. We propose that these short loops contain the DNA attachment sequences associated with the proteins of the LE. During the formation of the SC one of the two chromatin filaments incorporates at the central surface of the forming lateral element (LE) and the other is located at the external side of the LE. This unexpected distribution does not correspond to the pair of thick filaments previously discerned in structure of the LE. The presence of RNA associated with the DNA-containing thin filaments, as well as with the axial chromatin elements of the forming SC, may be related with the transcription occurring during meiotic prophase, specially during zygotene stage. We propose that RNA is involved in a still uncharacterized process essential for pairing

The synaptonemal complex (SC) is the central key structure for meiosis in organisms undergoing sexual reproduction. During meiotic prophase I, homologous chromosomes exchange genetic information at the time they are attached to the lateral elements by specific DNA sequences. Most of these sequences, so far identified, consist of repeat DNA, which are subject to chromatin structural changes during meiotic prophase I. In this work, we addressed the effect of altering the chromatin structure of repeat DNA sequences mediating anchorage to the lateral elements of the SC. Administration of the histone deacetylase inhibitor trichostatin A into live rats caused death of cells in the pachytene stage as well as changes in histone marks along the synaptonemal complex. The most notable effect was partial loss of histone H3 lysine 27 trimethylation. Our work describes the epigenetic landscape of lateral element-associated chromatin and reveals a critical role of histone marks in synaptonemal complex integrity.

The arrangement of compact chromatin of G0 lymphocytes was studied in three-dimensional reconstructions of the ensemble of the chromatin and of individual compact chromatin bodies. Rat spleen was serially cut and sections were contrasted with procedures preferential for DNA. Electron microscopy images were digitized, processed, and displayed using a commercial software package, complemented by a system for three-dimensional reconstruction and analysis developed by us on an IBM-compatible microcomputer provided with an image acquisition board. The reconstructions showed a continuous layer of compact chromatin in contact with the nuclear envelope that prevents the automatic recognition of individual chromatin clumps. The ensemble of the arrangement of compact chromatin was found to be very similar in different lymphocytes. After morphological filtering procedures, the initial mass was divided into individual bodies of compact chromatin, which were tagged. Most of these bodies contact the nuclear envelope. The number of bodies as well as the number of contacts with the envelope are similar and correspond to a haploid number of chromosomes. The largest body is always the one containing nucleolus-associated chromatin. When the cell has two nucleoli, the nucleolus-associated chromatin bodies contact the envelope in diametrically opposed areas. This feature was also described in rat liver cells. It is concluded that: (a) the individualized compact chromatin bodies do not correspond to an entire chromosome or to a pair of chromosomes; (b) the arrangement of compact chromatin is not identical in each G0 lymphocyte, but there are patterns that are repeated with limited changes; and (c) there are common features that appear in different cell types of individuals of the same species.