Transcriptional activity and splicing factors are preserved during physiological apoptosis.

Apoptosis is the best-known programmed cell death that maintains tissue homeostasis in eukaryotic cells. The morphological characteristics include nuclear and cytoplasmic contraction and cytoplasmic blebbing, its biochemical hallmarks include caspase protease activity and DNA fragmentation. In rat ovaries, cell death is a normal process that occurs throughout the organism's life. Granulosa cells, the more abundant cell type forming the ovarian follicles, are eliminated via different routes of cell death. Most granulosa cells are eliminated through apoptotic cell death. In this work, we analyzed the behavior of nuclear components throughout the apoptotic process and determined how they are regionalized and conserved during follicular atresia in rat ovaries. Apoptosis was detected based on caspase-3 activity and DNA fragmentation using the TUNEL technique. We identified the transcription markers H3ac and RNA Pol II, and splicing factor SC35 by immunodetection. The nucleolar components were analyzed via light microscopy and transmission electron microscopy through immunodetection of the proteins nucleolin and nucleophosmin-1. The nuclear ultrastructure was analyzed using standard contrast and preferential ribonucleoprotein contrast. Our results demonstrate that during the progression of apoptosis, chromatin is remodeled to constitute apoptotic bodies; transcription and spliceosome elements are reorganized along with the nucleolar components. Additionally, the splicing and transcription factors are segregated into specific territories inside the apoptotic bodies, suggesting that transcriptional elements are reorganized during the apoptotic process. Our results indicate that apoptotic bodies not only are compacted, and chromatin degraded but all the nuclear components are progressively reorganized during cell elimination; moreover, the transcriptional components are preserved.

Changes of the nucleolus architecture in absence of the nuclear factor CTCF.

CTCF is a multifunctional nuclear factor involved in many cellular processes like gene regulation, chromatin insulation and genomic organization. Recently, CTCF has been shown to be involved in the transcriptional regulation of ribosomal genes and nucleolar organization in Drosophila cells and different murine cell types, including embryonic stem cells. Moreover, it has been suggested that CTCF could be associated to the nucleolus of human erythroleukemic K562 cells. In the present work, we took advantage of efficient small hairpin RNA interference against human CTCF to analyze nucleolar organization in HeLa cells. We have found that key components of the nucleolar architecture are altered. As a consequence of such alterations, an upregulation of ribosomal gene transcription was observed. We propose that CTCF contributes to the structural organization of the nucleolus and, through epigenetic mechanisms, to the regulation of the ribosomal gene expression.

Analysis of different cell death processes of prepubertal rat oocytes in vitro.

The processes of cell death were studied in vitro in populations of oocytes isolated from prepubertal rats. In order to identify apoptosis, the externalized phosphatidylserine was recognized with Annexin-V coupled to FITC and the fragmentation of DNA was demonstrated by means of electrophoresis. Oocytes were tested for autophagy by means of the incorporation of monodansylcadaverine and monitoring Lc3-I/Lc3-II by western blot. The expression of mRNA marker genes of autophagy and of apoptosis was studied by means of RT-PCR in pure populations of oocytes. Some oocytes expressed at least one of the following markers: caspase-3, lamp1 and Lc3. Some oocytes were positive to Annexin-V or to monodansylcadaverine. However, most of them were simultaneously positive to both markers. The relative frequency of oocytes simultaneously positive to markers of apoptosis and autophagy did not change in the different ages studied. The transformation of Lc3-I in Lc3-II was present in all populations of oocytes studied. The mRNAs for caspase-3, lamp1 and Lc3 were present in all populations of oocytes analyzed. Our results demonstrate that oocytes of rats from new born to prepubertal age are eliminated by means of three different cell death processes: apoptosis, autophagy and a mixed event in which both routes to cell death participate in the same cell.

Chromatin structure contribution to the synaptonemal complex formation.

Meiosis is a key cellular and molecular process for sexual reproduction contributing to the genetic variability of organisms. This process takes place after DNA replication and consists in a double cellular division, giving rise to four haploid daughter cells or gametes. Meiotic recombination between homologous chromosomes, in the meiotic prophase I, is mediated by a tripartite structure named Synaptonemal Complex (SC). The SC is a peptidic scaffold in which the chromatin of homologous chromosomes is organized during the pachytene stage, holding chromosomes together until the meiotic recombination and genetic exchange have taken place. The role of chromatin structure in formation of the SC and the meiotic recombination at meiotic prophase I remain largely unknown. In this review we address the epigenome contribution to the SC formation at meiotic prophase I, with particular attention on the chromatin structure modifications occurring during the sub-stages of meiotic prophase I.

Combined apoptosis and autophagy, the process that eliminates the oocytes of atretic follicles in immature rats.

We studied the alterations of dying oocytes in 1-28 days old rats using TUNEL method, immunolocalizations of active caspase 3, lamp1, localization of acid phosphatase, and DAPI staining. All procedures were performed in adjacent sections of each oocyte. In most dying oocytes exist simultaneously features of apoptosis as active caspase 3 and DNA breaks, and a large increase of lamp1 and acid phosphatase characteristic of autophagy. Large clumps of compact chromatin and membrane blebbing were absent. Electron microscope observations demonstrated the presence of small clear vesicles and autophagolysosomes. All these features indicate that a large number of oocytes are eliminated by a process sharing features of apoptosis and autophagy. In dying oocytes of new born rats the markers of apoptosis predominate over those of autophagy. However, fragmentation and apoptotic bodies were not found. These features suggest that in different cytophysiological conditions the processes of cell death may be differently modulated.

Integration of morphological and cytophysiological studies on estrogen receptor.

Ultrastructural and immunocytochemical studies of an intra-nuclear particle, the perichromatin granule (PCG), demonstrated the presence of processed mRNA in this structure. Ovariectomy caused an increase in the number of PCGs in uterine cells and administration of estradiol drastically reduced the nuclear pool of PCGs in 15 min. In vitro studies demonstrated that this depletion was accompanied by an increase of the export of previously synthesized RNA. Similar quantitative changes of the abundance of PCG and of the rate of the export of RNA were found in ventral prostate after orchiectomy and testosterone restitution, as well as in the target cells of FSH, LH, TSH, and ACTH. These results taken together led us to conclude that PCGs constitute an intra-nuclear compartment of a few processed mRNA in equilibrium with transcription and export. This mRNA is rapidly transferred to the cytoplasm by specific hormone signals.

Ultrastructural and autoradiographic study of the effects of bleomycin on the interphase nucleus of cultured normal cells.

Primary cultures of hepatocytes and epithelial endometrial cells were treated with bleomycin (10 to 200 microgram/ml) for 30 to 300 min. Structural changes were studied with a staining method which contrasts ribonucleoproteins. The earliest visible alteration was the accumulation of perichromatin granules in association with the nucleolus. This disturbance was frequently accompanied by modifications in the nucleolar architecture. After larger treatments, the most striking changes were nucleolar segregation and the appearance of spherical clear bodies in the nucleolus. In the extranucleolar area, a remarkable diminution of ribonucleoprotein fibrils and clustering of interchromatin granules were observed. Functional disturbances in the synthesis and transporting of RNA to the cytoplasm were studied by high-resolution quantitative autoradiography after labeling with tritiated uridine. Bleomycin produces a strong inhibition of RNA synthesis in nucleolar and extranucleolar areas. Important decreases of [3H]uridine incorporation were observed as early as 30 min after the administration of drug. Alterations of processing and/or transporting of RNA to the cytoplasm were found after treatments with bleomycin (100 microgram/ml) for 30 to 300 min. It is suggested that the diminution of ribonucleoprotein fibrils is related to the inhibition of RNA synthesis while the accumulation of perichromatin granules is connected to alteration of the transporting and/or processing.

Immunocytochemical study of estrogen receptor activation factor (E-RAF) and the proteins that interact with nuclear estrogen receptor II (nER II) in epithelial endometrial cells, in the presence and in the absence of estradiol.

The localization and abundance of the estrogen receptor activation factor (E-RAF) and a small nuclear ribonucleoprotein (snRNP) complex containing three proteins, p32, p55 and p60, which interact with the nuclear estrogen receptor II (nER II), have been studied in rat endometrial epithelial cells by means of immunofluorescence and high resolution quantitative immunocytochemistry. In the cytoplasm E-RAF is associated with the rough endoplasmic reticulum. In the nucleus it is mainly localized at the interchromatin space, and surrounding the clumps of compact or semi-condensed chromatin. Quantitative analyses show that the abundance of E-RAF in the nucleus increases after ovariectomy and decreases 3 minutes after estradiol administration. These results are in agreement with the currently available biochemical data. Double immunolocalizations demonstrate that p32, p55, p60 co-localize with other splicing-related protein. High resolution immunolocalization shows that p32, p55, p60 are associated with perichromatin fibrils (co-transcriptional splicing) and with clusters of interchromatin granules (storage of splicing-related molecules). The nuclear abundance of the snRNP complex decreases with ovariectomy, increases within 3 minutes after estradiol administration and remains higher than that in ovariectomized animals for 27 minutes. These results strongly support the previous data on the role of nER-II in the regulation of mRNA transcription and its export from the nucleus to the cytoplasm.

Immunohistochemical and ultrastructural study of the lamellae of oocytes in atretic follicles in relation to different processes of cell death.

Atresia is the process through which non-selectable oocytes are eliminated; it involves apoptosis and/or autophagy. This study used immunohistochemical and ultrastructural techniques to characterize the lamellae present in the cytoplasm of oocytes in follicles in the process of atresia in prepubertal and adult Wistar rats. The results indicate that the lamellae are positive to tubulin and myosin immunodetection under light and electron microscopy. Labeling is greater with anti-tubulin and lesser with anti-myosin. Our observations indicate that lamellae are present in oocytes at the initial antral stage in prepubertal rats; that is, from day 14 post-birth to adult age. We were able to determine that the increase in altered lamellae principally occurs in the apoptotic cells rather than in the autophagic cells.

Immunocytochemical characterization of nuclear ribonucleoprotein fibrils in cells of the central nervous system of the rat.

Nucleoplasmic structural constituents observed in partially decondensed nuclei of the central nervous system of the rat were analyzed by postembedding immunoelectron microscopy using antibodies specifically recognizing heterogenous nuclear ribonucleoprotein (hnRNP) and small nuclear ribonucleoprotein (snRNP) complexes and DNA. Fibrogranular RNP structures (polyparticles) were found in close proximity to DNA containing fibrillar areas resulting from partial dispersion of compact chromatin. The polyparticle-type fibrils are labeled by antibodies recognizing hnRNP core proteins as well as snRNPs (Sm antigen or 70 kDa protein of U1snRNP) or the m3G-cap structure of snRNAs. These observations suggest that such polyparticle-type fibrils correspond to extended perichromatin fibrils. Partially decondensed perichromatin granules are rarely labeled by anti-snRNP or snRNA antibodies. When labeling occurs it is restricted to the periphery of the granules. However, anti-hnRNP antibodies frequently label these granules. Our results favor the idea, previously proposed for Balbiani ring granules, that perichromatin granules are formed by the folding of hnRNP containing perichromatin fibrils (polyparticles) in the process of splicing, and that mature perichromatin granules contain already spliced messenger RNA.

Neuronal distribution in caudate nucleus and reticular-caudate pathways.

Methods of quantitative stereology are employed to determine the cell body diameters and the disposition of the neurons of caudate nucleus (CN) of the cat. Large neurons are more frequent in the infero-external region in a well defined zone 16 to 18 mm ahead of the interaural plane. Silver impregnations and electron microscopy after lesions in the ponto-mesencephalic reticular formation demonstrate its direct projections on the nucleus centralis medialis thalami and on the CN. Ascending fibers run along the ventral and lateral surfaces of the thalamus, some of them penetrate to the lamina medullaris medialis making contact in intralaminar nuclei and in the nucleus centralis medialis, others continue through the internal capsule to end in the infero-external region of the CN. The reticular formation of one side projects to both CN.

A simple staining method for chromatin in electron microscopy compatible with serial sectioning.

The study of the disposition of chromatin in the interphasic nucleus requires the combination of serial sectioning and a specific or preferential chromatin staining. The staining of chromatin with phosphotungstic acid (PTA) chromatin was originally employed on sections of glycolmethacrylate-embedded samples. As it is very difficult to obtain ribbons with this resin, we introduced a modification which consisted of staining the tissue after fixation and before dehydration, in order that epoxy resins can be applied. Several procedures were tried and the best results were attained in the following way. Standard fixation of samples no thicker than 1 mm was carried out with 2.5% glutaraldehyde at pH 7.2 for 1 or 2 h at room temperature; tissues were then rinsed three times with 0.2N HCl adjusted to pH 2.1-2.3 with 0.2N NaOH for 15 min. Staining was held with 3% W/V PTA in 1N HCl adjusted to the same pH. Samples were dehydrated in gradual ethyl alcohol concentrations and Epon-embedded. Post-staining on sections with uranyl-acetate and lead-citrate or other methods may be used to demonstrate other cell components and their relations to chromatin.

Implications for evolution of nuclear structures of animals, plants, fungi and protoctists.

The evolutionary variations of nuclear structure of animals, plants, fungi and protoctists were studied with electron microscopy by using techniques preferentially staining ribonucleoprotein (RNP) particles and chromatin. A remarkable similarity in the general morphological features of the RNP particles and chromatin arrangement is found in animals, plants and fungi. Important variations of these features were found in protoctists. These observations suggest that major evolutionary changes in the nuclear structure predate the acquisition of plastids by the ancestors of green plants. Once evolved, the nuclear structural pattern is conserved in plants and animals. Among protoctists studied, Kinetoplastida, Cryptomonadida and Volvocida have RNP particles and chromatin arrangement resembling those of plants and animals. These similarities may indicate a common ancestor. Important differences in the nuclear structure among Euglenida, Amebida, Cryptomonadida, Volvocida and Kinetoplastida support the view that Sarcomastigophora is a polyphyletic taxon. For the same reason Kinetoplastida and Euglenida must not be grouped in a monophyletic taxon. We propose that the variations of RNP particles may be related to the initial evolution of post-transcriptional processing.

Activation of osmium ammine by SO2-generating chemicals for EM Feulgen-type staining of DNA.

We present herein an improved method for the use of osmium ammine in a Feulgen-type reaction for specific staining of DNA at EM level and an analysis of its Schiff-type reagent behaviour. The activation of osmium ammine to a Schiff-type reagent (so far routinely performed by bubbling with gaseous SO2) can be accomplished by adding S0(2)-generating chemicals as for light microscopy. When used after HCI hydrolysis on epon or acrylate sections, activated osmium ammine behaves like a Schiff-type reagent, and the DNA staining can be selectively and completely abolished by aldehyde blocking agents. This preparation has the advantage of eliminating the use of gaseous SO2 thus rendering the technique more widely available to laboratories which cannot handle gas cylinders containing SO2. We recommend the use of osmium ammine in 8N acetic acid and 40mM sodium metabisulfite for 1 h at 37 degrees C for epon sections, and in 0.2N HCl and 0.2M metabisulfite for 30 min at room temperature for acrylate sections.

Ultrastructural and immunocytochemical analysis of the XY body in rat and Guinea pig.

The formation of the XY body involves the compaction of the extended chromatin to form a mesh of fibrogranular structures. During this process the ribonucleoprotein particles (RNP), which were associated with the chromatin filaments progressively disappear. High resolution immunolocalization indicates that the mature XY body does not contain RNA polymerase II, hnRNPs, or snURNPs. Occasionally chromatin fibrils extend outside of the XY body. These fibrils are frequently associated with nascent RNP fibrils and granules indicating that not all the DNA of the sex chromosomes is transcriptionally inactive. However, transcription is located outside the sex body. The recombination protein Dmc1 is present in nodules associated with the unpaired chromosomal axes of the sex chromosomes located in the XY body. Cytochemical staining methods and in situ hybridization at electron microscopic level show that RNA is present in the unpaired chromosomal axes suggesting that the presence of RNA in the chromosomal axes and in forming synaptonemal complexes is related with the process of final pairing. The sex body and the nucleoli associated with it do not interweave and do not exchange RNA or DNA-containing filaments. These observations indicate that the spatial relation between these structures is just a close proximity, which is, however, very frequent.

Cytochemical study of the distribution of RNA and DNA in the synaptonemal complex of guinea-pig and rat spermatocytes.

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.

[Ultrastructural and immunocytochemical analysis of the synaptonemal complex at the initiation of synapsis]. / Análisis ultraestructural e immunocitoquímico del complejo sinaptonémico al inicio de la sinapsis.

The synaptonemal complexes (SCs) are nuclear structures specific for meiosis. They have a central role in homolog chromosomes coupling; they are essential in crossing over events and chromosomic segregation during the first meiotic division. When its joining ends in pakiteno stage, each synaptonemal extends along the bivalent joining the ends to nuclear wrapping. The SCs are characterized by the presence of two lateral elements and a central region. The lateral elements are parallel and equidistant. The chromatine of homolog chromosomes fixes in a series of loops to these elements. The central region is between the lateral elements. It is formed by the latero-medial fibers and the medial element. The first ones are perpendicularly oriented to the longitudinal axis of CS and connect lateral elements with the medial element. The recombination modules have an active role in recombination processes and quiasma formation, they are associated, at intervals, with the central region among the homolog chromosomes. The localization and function of nucleic acids in formation and coupling of synaptonemal complex is little known, so methodologic alternatives are looked for to resolve this type of problems. In this work, ADN distribution in chicken ovocytes in cigotene, using techniques for electronic microscopy of immuno-oro, were studied. Besides, cytochemical techniques, were used as preferential contrast for ADN or preferential for ribonucleoproteins (RNPs). The combination of preferential tincture for RNPs and immunolocalization of ADN show that chromatin accumulates jointly with ribonucleoproteins in nor coupled lateral elements and the presence of numerous RNPs fibers distributed around lateral elements. Recombination nodules were found among lateral elements during the coupling, these nodules are PTA positives, which means ADN presence, and so, ADN presence among lateral elements. THe presence of a bridge of marked fibers with coloidal gold (ADN) uniting not coupled lateral elements, suggests ADN as a sort of macromollecule forming synapsis sites.

Immunohistochemical and ultrastructural visualization of different routes of oocyte elimination in adult rats.

Cell death is a process for maintaining homeostasis in tissues and organs. In the ovary, apoptotic cell death has been implicated in follicular atresia; in the elimination of the follicles that are not ovulated during adult life. Recent studies indicate that apoptosis and autophagy are two programmed processes of cell death. Apoptosis is performed by proteases called caspases and leads to such morphological traits as DNA fragmentation. Autophagy, in turn, is characterized by the exacerbated formation of autophagosomes; a process in which the amount of the LC3 and Lamp 1 proteins increases. In this study, oocytes from all stages of the estrous cycle of Wistar rats were analyzed. The apoptosis process was identified by immunodetecting active Caspase-3 and locating DNA fragmentation using the TUNEL technique. Autophagy was evaluated through immunodetection of the LC3 and Lamp 1 proteins, and by ultrastructural localization of autophagic vesicle formation. All techniques were conducted using the same oocytes. Results show that all phases of the estrous cycle contain dying oocytes that test positive simultaneously for apoptosis and autophagy markers. The highest level of apoptosis was found during estrus; while the proestrous stage had the highest level of autophagy. The diestrous and metestrous phases were characterized by a high frequency of the presence of markers of apoptosis and autophagy in the same oocyte. Our results demonstrate that during oocyte elimination in adult rats the proteins involved in both processes, apoptosis and autophagy, are present in the same cell at the same time.