Pattern Of Chromatin Condensation Research Articles

The coasting time affects the quality of cumulus-oocyte complexes in superstimulated ewes

We hypothesized that the coasting time may be beneficial to the quality of cumulus-oocyte complexes recovered from live ewes, as reported in cattle. The present study assessed the effect of coasting times on the quantity and quality of cumulus-oocyte complexes (COCs) in sheep. All ewes were subjected to the “Day 0 protocol”, followed by an ovarian stimulation (80 mg of pFSH in three decreasing doses), varying only the coasting time [12 (G12), 36 (G36), or 60 h (G60]. In Experiment 1, data regarding follicular population was assessed. In Experiment 2, the COC quality was checked by their morphology, brilliant cresyl blue (BCB) test, evaluation of chromatin condensation pattern, and oocyte diameter. In Experiment 3, genes related to oocyte developmental competence were evaluated in BCB + COCs. The oocytes in the G60 group had more (P < 0.05) large follicles than the other groups and oocytes with a greater diameter than the G12. Oocyte morphology was similar (P > 0.05) among groups, as well as the BCB + COCs quantity. The G60-oocytes presented a better (P < 0.05) configuration of chromatin condensation compared with the other groups and a greater (P < 0.05) gene expression of BMP15, MATER, ZAR1, and PTGS2 compared with G12, and PTGS2 and HAS2 compared with G36 group. In conclusion, 60 h of coasting time positively affects the quality of COCs recovered after subjecting ewes to the “Day 0 protocol” and ovarian superstimulation. Implementing the appropriate coasting time to a given protocol can positively impact the in vitro embryo production outcomes in sheep.

Diversity of chromatin condensation patterns, nuclear reorganization, evolution and phylogenetic distribution of sperm nuclear basic proteins in fish

Diversity of chromatin condensation patterns, nuclear reorganization, evolution and phylogenetic distribution of sperm nuclear basic proteins in fish

Spermiogenic chromatin condensation patterning in several hexapods may involve phase separation dynamics by spinodal decomposition or microemulsion inversion (nucleation)

We have examined published transmission electron microscopy (TEM).photomicrographs of chromatin condensation patterning in developing sperm nuclei from five species of entognathous hexapods within the Classes Protura, Collembola, Diplura and five species of ancestral wingless insects in the Orders Archaeognatha and Zygentoma as well as in fifteen species of the winged insects. Each species reproduces by internal fertilization. Spatially quantitative analysis indicates that spermiogenic chromatin condensation patterning in several of these species may be due to spinodal decomposition (SD) or to microemulsion inversion (chromatin-in-nucleoplasm → nucleoplasm-in-chromatin), also known as nucleation (Nc). These are two different dynamic mechanisms of liquid–liquid phase separation (LLPS). They might either occur independently or co-exist during the chromatin condensation associated with insect spermiogenesis. For example, the chromatin condensation pattern such as that observed in transverse sections of developing sperm nuclei from the wingless insect Anurida maritima (Collembola) is: granules → fibers → lamellae (SD) → nucleation (Nc) → condensed nuclei. Similar transitions are also observed in other more recently evolved species within the Class Insecta. From the limited but comprehensive sample of entognathus and ectognathus hexapods analyzed here, it appears that LLPS of sperm chromatin during spermiogenesis has occurred quite pervasively within the subphylum Hexapoda, including insects.

Trypan Blue - Adapting a Dye Used for Labelling Dead Cells to Visualize Pinocytosis in Viable Cells.

Trypan blue is routinely used in cell culture experiments to distinguish between dead cells, which are labelled by trypan blue, and viable cells, which are apparently free of any staining. The assumption that trypan blue labelling is restricted to dead cells derives from the observation that rupture of the plasma membrane correlates with intense trypan blue staining. However, decades ago, trypan blue has been used to trace fluid uptake by viable macrophage-like cells in animals. These studies contributed to the concept of the reticuloendothelial system in vertebrates. Trypan blue itself does not show a fluorescence signal, but trypan blue-labelled proteins do. Therefore, intracellular localization of trypan blue-labelled proteins could give a clue to the entrance pathway of the dye in viable cells. We used fluorescence microscopy to visualize trypan blue positive structures and to evaluate whether the bactericide, silver, enhances cellular trypan blue uptake in the brain macrophage-like cell line, BV-2. The pattern of chromatin condensation, visualized by DAPI staining, was used to identify the cell death pathway. We observed that silver nitrate at elevated concentrations (≥ 10 µM) induced in most cells a necrotic cell death pathway. Necrotic cells, identified by pycnotic nuclei, showed an intense and homogenous trypan blue staining. Apoptotic cells, characterized by crescent-like nuclear chromatin condensations, were not labelled by trypan blue. At lower silver nitrate concentrations, most cells were viable, but they showed trypan blue labelling. Viable cells showed a cell-type specific distribution of heterochromatin and revealed a perinuclear accumulation of bright trypan blue-labelled vesicles and, occasionally, a faint homogenous trypan blue labelling of the cytoplasm and nucleus. Amiloride, which prevents macropinocytosis by blocking the Na+ / H+ exchange, suppressed perinuclear accumulation of dye-labelled vesicles. Swelling of cells in a hypotonic solution induced an intense intracellular accumulation of trypan blue. Cells exposed to a hypotonic solution in the presence of 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), which blocks volume-regulated ion channels, prevented labelling of the cytoplasm and nucleus but did not affect labelling of perinuclear vesicles. In viable cells trypan blue-labelled vesicles indicate trypan blue uptake by macropinocytosis and trypan blue-labelled cytosol could indicate a further entry pathway for the dye, like activated volume-regulated channels. Accordingly, fluorescence microscopic analysis of trypan blue-labelled cells allows not only a discrimination between necrotic and apoptotic cell death pathway but also a discrimination between the mode of trypan blue uptake in viable cells - via pinocytosis or via activated volume-regulated ion channels - in the same preparation at the single cell level.

Corrigendum] Cyclophilin D modulates cell death transition from early apoptosis to programmed necrosis induced by honokiol

Subsequently to the publication of the above article, the authors have realized that the printed version of Fig.6 on p.1661 contained some mistakes. Potential anomalies regarding this figure concerning the duplication of data both within Fig.6 and comparing data between Figs.5 and 6 were also drawn to our attention by an interested reader. Specifically, the authors realized that the bands of BCL‑xl were erroneously selected from the GAPDH dataset during the process of compiling this figure. The authors subsequently found that the original photographs of these western blot bands had been lost during the time period that had elapsed since these experiments were completed. In order to corroborate the results, the authors repeated the contentious experiments shown in Fig.6 and obtained similar results, thereby corroborating the results and conclusions reported in this study. A revised version of Fig.6, containing the newly obtained data, is shown below. The errors made with the assembly of Fig.6 originally did not have an adverse bearing on the overall conclusions reported in this study. The authors are grateful to the Editor of International Journal of Oncology for allowing them the opportunity to publish this Corrigendum, and all of the authors agree to the publication of this Corrigendum. The authors sincerely apologize for their errors, and apologize to the readership of the Journal for any inconvenience caused. [the original article was published in International Journal of Oncology 42:1654‑1663, 2013; DOI: 10.3892/ijo.2013.1863].

Histone deacetylases inhibitors as new potential drugs against Leishmania braziliensis, the main causative agent of new world tegumentary leishmaniasis

The protozoan parasite Leishmania braziliensis is a major causative agent of the neglected tropical diseases Cutaneous and Mucocutaneous Leishmaniases in the New World. There are no vaccines to prevent the infection and the treatment relies on few drugs that often display high toxicity and costs. Thus, chemotherapeutic alternatives are required. Histone Deacetylases (HDACs) are epigenetic enzymes involved in the control of chromatin structure. In this work, we tested an in-house library of 78 hydroxamic acid derivatives as putative inhibitors of L. braziliensis HDACs (HDACi). The compounds were evaluated in relation to the toxicity to the host cell macrophage and to the leishmanicidal effect against L. braziliensis during in vitro infection. Eight HDACi showed significant leishmanicidal effects and the top 5 compounds showed effective concentrations (EC50) in the range of 4.38 to 10.21 μM and selectivity indexes (SI) from of 6 to 21.7. Analyses by Transmission Electron Microscopy (TEM) indicated induction of apoptotic cell death of L. braziliensis amastigotes with a necrotic phenotype. An altered chromatin condensation pattern and cellular disorganization of intracellular amastigotes was also observed. A tight connection between the mitochondrion and nuclear protrusions, presumably of endoplasmic reticulum origin, was found in parasites but not in the host cell. In flow cytometry (FC) analyses, HDACi promoted parasite cell cycle arrest in the G2-M phase and no changes were found in macrophages. In addition, the direct effect of HDACi against the promastigotes showed apoptosis as the main mechanism of cell death. The FC results corroborate the TEM analyses indicating that the HDACi lead to changes in the cell cycle and induction of apoptosis of L. braziliensis. The production of nitric oxide by the infected macrophages was not altered after treatment with the top 5 compounds. Taken together, our results evidenced new HDACi as promising agents for the development of new treatments for American Tegumentary Leishmaniasis caused by L. braziliensis.

Predicting cell lineages using autoencoders and optimal transport.

Lineage tracing involves the identification of all ancestors and descendants of a given cell, and is an important tool for studying biological processes such as development and disease progression. However, in many settings, controlled time-course experiments are not feasible, for example when working with tissue samples from patients. Here we present ImageAEOT, a computational pipeline based on autoencoders and optimal transport for predicting the lineages of cells using time-labeled datasets from different stages of a cellular process. Given a single-cell image from one of the stages, ImageAEOT generates an artificial lineage of this cell based on the population characteristics of the other stages. These lineages can be used to connect subpopulations of cells through the different stages and identify image-based features and biomarkers underlying the biological process. To validate our method, we apply ImageAEOT to a benchmark task based on nuclear and chromatin images during the activation of fibroblasts by tumor cells in engineered 3D tissues. We further validate ImageAEOT on chromatin images of various breast cancer cell lines and human tissue samples, thereby linking alterations in chromatin condensation patterns to different stages of tumor progression. Our results demonstrate the promise of computational methods based on autoencoding and optimal transport principles for lineage tracing in settings where existing experimental strategies cannot be used.

Nuclear morphometrics and chromatin condensation patterns as disease biomarkers using a mobile microscope.

Current cancer diagnosis involves the use of nuclear morphology and chromatin condensation signatures for accurate advanced stage classification. While such diagnostic approaches rely on high resolution imaging of the cell nucleus using expensive microscopy systems, developing portable mobile microscopes to visualize nuclear and chromatin condensation patterns is desirable at clinical settings with limited infrastructure. In this study, we develop a portable fluorescent mobile microscope capable of acquiring high resolution images of the nucleus and chromatin. Using this we extracted nuclear morphometric and chromatin texture based features and were able to discriminate between normal and cancer cells with similar accuracy as wide-field fluorescence microscopy. We were also able to detect subtle changes in nuclear and chromatin features in cells subjected to compressive forces, cytoskeletal perturbations and cytokine stimulation, thereby highlighting the sensitivity of the portable microscope. Taken together, we present a versatile platform to exploit nuclear morphometrics and chromatin condensation features as physical biomarkers for point-of-care diagnostic solutions.

Testicular structure and spermatogenesis of short mackerel, Rastrelliger brachysoma (Bleeker, 1851) in Upper Gulf of Thailand

Testicular structure and spermatogenesis of short mackerel, Rastrelliger brachysoma (Bleeker, 1851) (Teleostei: Scombridae) was first investigated. The testicular parenchyma was a lobular organ, which was classified as an unrestricted spermatogonial type. The classification of spermatogenetic stage could be classified into six stages based on the pattern of chromatin condensation and other characterizations at the light microscopic level. These six stages included the primary and secondary spermatogonium, primary and secondary spermatocyte, spermatid and spermatozoon. The spermatogenesis could also be classified into another four stages based on the nuclear and cytoplasmic characterizations at the ultrastructural level. Spermatogonium was the early germ cell. It underwent a series of mitotic division to reach the primary spermatocyte. Secondary spermatocyte was shown as the heterochromatin surrounding the nuclear membrane, which was rarely seen within seminiferous lobules. Stages during the spermatids differentiation comprised of the early, intermediate and late stages which are under the degree/change of chromatin condensation. Finally, the spermatozoon was revealed as the aquasperm primitive type. It was composed of an oval head without an acrosome, a short mid-piece consisting of two basal bodies (proximal and distal centrioles) and a long flagella tail without lateral fins. The axonemes of classical form with 9+2 microtubules were presented in the flagellum.

Interpopulation hybridization generates meiotically stable rDNA epigenetic variants in allotetraploid Tragopogon mirus.

Uniparental silencing of 35S rRNA genes (rDNA), known as nucleolar dominance (ND), is common in interspecific hybrids. Allotetraploid Tragopogon mirus composed of Tragopogon dubius (d) and Tragopogon porrifolius (p) genomes shows highly variable ND. To examine the molecular basis of such variation, we studied the genetic and epigenetic features of rDNA homeologs in several lines derived from recently and independently formed natural populations. Inbred lines derived from T. mirus with a dominant d-rDNA homeolog transmitted this expression pattern over generations, which may explain why it is prevalent among natural populations. In contrast, lines derived from the p-rDNA dominant progenitor were meiotically unstable, frequently switching to co-dominance. Interpopulation crosses between progenitors displaying reciprocal ND resulted in d-rDNA dominance, indicating immediate suppression of p-homeologs in F1 hybrids. Original p-rDNA dominance was not restored in later generations, even in those segregants that inherited the corresponding parental rDNA genotype, thus indicating the generation of additional p-rDNA and d-rDNA epigenetic variants. Despite preserved intergenic spacer (IGS) structure, they showed altered cytosine methylation and chromatin condensation patterns, and a correlation between expression, hypomethylation of RNA Pol I promoters and chromatin decondensation was apparent. Reversion of such epigenetic variants occurred rarely, resulting in co-dominance maintained in individuals with distinct genotypes. Generally, interpopulation crosses may generate epialleles that are not present in natural populations, underlying epigenetic dynamics in young allopolyploids. We hypothesize that highly expressed variants with distinct IGS features may induce heritable epigenetic reprogramming of the partner rDNA arrays, harmonizing the expression of thousands of genes in allopolyploids.

Spermatogenesis in the rock oyster, Saccostrea forskali (Gmelin, 1791).

Morphology of the differentiating spermatogenic cells of the rock oyster Saccostrea forskali (Bivalve: Ostreidae) was investigated by light and transmission electron microscopy. The testis is formed by several branching acini containing developing spermatogenic cells, classified into 7 stages based on nuclear characteristics, patterns of chromatin condensation and cytoplasmic contents. The spermatogonium is characterized by a euchromatic nucleus with a prominent nucleolus. The cytoplasm contains several round granulo-fibrillar dense bodies surrounded by numerous mitochondria. The round nucleus of the primary spermatocyte contains patches of electron-dense heterochromatin, numerous proacrosomal vesicles, ribosomes and mitochondria. The secondary spermatocytes contain a reticulated chromatin pattern and reduced number of proacrosomal vesicles. The early spermatids contain a small amount of euchromatin among dense patches of heterochromatin. A large single acrosomal vesicle is located in the posterior part of the cell. The middle spermatid is characterized by the migration of an acrosomal vesicle to the anterior part of the nucleus. The late spermatids contain highly condensed heterochromatin blocks and the acrosomal vesicle becomes cup-shaped and invaginated at the basal part. The spermatozoon contains a barrel-shaped head covered with the cup-like acrosome. At this stage, the subacrosomal space contains an axial rod in subacrosomal materials. Three to four transverse bands appear at the anterior region of the acrosome. The middle piece consists of spherical mitochondria surrounding the proximal and distal centrioles. The flagellum consists of 9+2 axonemal microtubule doublets surrounded by the plasma membrane. Our electron microscopic study of spermatogenesis in the S. forskali provides important new information on the mechanism of development of spermatogenesis of this species.

First successful pregnancy in a globozoospermic patient having homozygous mutation in SPATA16

To report the first case of a successful pregnancy obtained with an infertile 29-year-old man with a homozygous mutation in the spermatogenesis-specific gene SPATA16 after intracytoplasmic sperm injection (ICSI). Case report. Assisted reproductive technology (ART) center. A couple who suffered from infertility of whom the husband was globozoospermic and carrying a homozygous mutation in the spermatogenesis-specific gene SPATA16. Clinical and histopathological evaluation, transmission electron microscopy, stimulation with fixed antagonist protocol, ICSI, activation of fertilization. Invitro fertilization and pregnancy. Oligoasthenoteratozoospermia was revealed in semen analysis. Total globozoospermia was detected. Chromatin condensation pattern was detected abnormal in 88% of spermatozoa with aniline blue staining. During the ART cycle 12 oocytes were collected. Eleven oocytes were at the metaphase II and one was at the germinal vesicle stage. After the injection, one oocyte fertilized on the following day; the 4-cell stage embryo was transferred. Pregnancy occurred and a healthy boy was delivered after 42weeks of gestation. This is the first report of a pregnancy obtained in a man with homozygous SPATA16 mutated globozoospermia.

The Calpain Inhibitor MDL28170 Induces the Expression of Apoptotic Markers in Leishmania amazonensis Promastigotes

BackgroundHuman cutaneous leishmaniasis is caused by distinct species, including Leishmania amazonensis. Treatment of cutaneous leishmaniasis is far from satisfactory due to increases in drug resistance and relapses, and toxicity of compounds to the host. As a consequence for this situation, the development of new leishmanicidal drugs and the search of new targets in the parasite biology are important goals.Methodology/Principal FindingsIn this study, we investigated the mechanism of death pathway induced by the calpain inhibitor MDL28170 on Leishmania amazonensis promastigote forms. The combined use of different techniques was applied to contemplate this goal. MDL28170 treatment with IC50 (15 µM) and two times the IC50 doses induced loss of parasite viability, as verified by resazurin assay, as well as depolarization of the mitochondrial membrane, which was quantified by JC-1 staining. Scanning and transmission electron microscopic images revealed drastic alterations on the parasite morphology, some of them resembling apoptotic-like death, including cell shrinking, surface membrane blebs and altered chromatin condensation pattern. The lipid rearrangement of the plasma membrane was detected by Annexin-V labeling. The inhibitor also induced a significant increase in the proportion of cells in the sub-G0/G1 phase, as quantified by propidium iodide staining, as well as genomic DNA fragmentation, detected by TUNEL assay. In cells treated with MDL28170 at two times the IC50 dose, it was also possible to observe an oligonucleossomal DNA fragmentation by agarose gel electrophoresis.Conclusions/SignificanceThe data presented in the current study suggest that MDL28170 induces apoptotic marker expression in promastigotes of L. amazonensis. Altogether, the results described in the present work not only provide a rationale for further exploration of the mechanism of action of calpain inhibitors against trypanosomatids, but may also widen the investigation of the potential clinical utility of calpain inhibitors in the chemotherapy of leishmaniases.

Cyclophilin D modulates cell death transition from early apoptosis to programmed necrosis induced by honokiol

Honokiol is a pharmacologically active small molecule with multifunctional antitumor effects. Although plenty of literature is available on honokiol-triggered apoptosis and programmed necrosis, few studies have investigated the potential existence of death mode transition from apoptosis to programmed necrosis. In the current study, we demonstrated that the necrotic cell population (PI-positive) gradually increased and the early-stage apoptotic cell population (PI-negative and AV-positive) decreased in a dose- and time-dependent manner following honokiol treatment. Furthermore, we demonstrated that these PI-positive cells were under necrotic cell death, since no late-apoptosis characteristics including conspicuous chromatin condensation or DNA ladder patterns were detected. These results demonstrated that cells suffered death mode transition from early-stage apoptosis to programmed necrosis with the increase of honokiol dose or treatment time. The protein expression of RIP3 markedly increased in parallel with HNK-triggered death mode transition, while the expression of RIP1 decreased. Cyclophilin D expression increased during cell death mode transition, and inhibition of cyclophilin D by cyclosporin A clearly blocked HNK-triggered programmed necrosis. These data indicated that honokiol-induced programmed necrosis and death mode transition are potentially RIP3‑dependent, cyclophilin D-regulated. Further results showed that blocked cyclophilin D by cyclosporin A inhibited HNK-induced necrosis, but did not affect HNK-induced RIP3 overexpression. This indicated that cyclophilin D was a potential modulator at downstream of RIP3. In conclusion, honokiol triggers a potential RIP3-dependent cell death mode transition from early-stage apoptosis to programmed necrosis, which is highly regulated by cyclophilin D.

Complex chromatin condensation patterns and nuclear protein transitions during spermiogenesis: Examples from mollusks

In this paper we review and analyze the chromatin condensation pattern during spermiogenesis in several species of mollusks. Previously, we had described the nuclear protein transitions during spermiogenesis in these species. The results of our study show two types of condensation pattern: simple patterns and complex patterns, with the following general characteristics: (a) When histones (always present in the early spermatid nucleus) are directly replaced by SNBP (sperm nuclear basic proteins) of the protamine type, the spermiogenic chromatin condensation pattern is simple. However, if the replacement is not direct but through intermediate proteins, the condensation pattern is complex. (b) The intermediate proteins found in mollusks are precursor molecules that are processed during spermiogenesis to the final protamine molecules. Some of these final protamines represent proteins with the highest basic amino acid content known to date, which results in the establishment of a very strong electrostatic interaction with DNA. (c) In some instances, the presence of complex patterns of chromatin condensation clearly correlates with the acquisition of specialized forms of the mature sperm nuclei. In contrast, simple condensation patterns always lead to rounded, oval or slightly cylindrical nuclei. (d) All known cases of complex spermiogenic chromatin condensation patterns are restricted to species with specialized sperm cells (introsperm). At the time of writing, we do not know of any report on complex condensation pattern in species with external fertilization and, therefore, with sperm cells of the primitive type (ect-aquasperm). (e) Some of the molluskan spermiogenic chromatin condensation patterns of the complex type are very similar (almost identical) to those present in other groups of animals. Interestingly, the intermediate proteins involved in these cases can be very different. In this study, we discuss the biological significance of all these features and conclude that the appearance of precursor (intermediate) molecules facilitated the development of complex patterns of condensation and, as a consequence, a great diversity of forms in the sperm cell nuclei.

Characterization of an Alternative Chromatin Remodeling to Parasperm in a Cottid Fish,Hemilepidotus gilberti

The dimorphic sperm of Hemilepidotus gilberti, i.e., haploid eusperm and diploid parasperm, have different morphologies corresponding to their own roles in fertilization. To estimate how these specific sperm morphologies were established, we focused on the nuclear morphologies and examined their changing processes in dimorphic spermiogenesis. Electron microscopic observation revealed that, in euspermatids, chromatin condensation first appeared as a mosaic pattern of moderate electrodense material in the peripheral region of the round nucleus. Those materials spread across the whole area to form a uniformly condensed nucleus. Chromatin condensation began similarly in paraspermatids to that in euspermatids. These became localized to one side of a nucleus and further condensed to form strong electrodense chromatin clusters, which are a specific feature of parasperm. From the remodeled nuclei of eusperm and parasperm, we found five and three kinds of sperm-specific basic proteins (SBPs), respectively, substituted to histones. The N-terminus amino acid sequences of the SBPs suggest that, in parasperm, one major SBP and two minor ones were distinct from each other. In eusperm nuclei, two kinds of specific SBPs were detected in addition to the homologs of parasperm SBPs. The specific SBPs had homologous amino acid sequences with huge arginine clusters, and one of them was most dominant among the five kinds of SBPs. The different combinations of SBPs in the eusperm and parasperm may cause a specific pattern of chromatin condensation in the dimorphic sperm nuclei of H. gilberti.

Cell fixation in zinc salt solution is compatible with DNA damage response detection by phospho‐specific antibodies

By virtue of superior preservation of proteins and nucleic acids the zinc salt-based fixatives (ZBF) has been proposed as an alternative to precipitants and cross-linking fixatives in histopathology. It was recently reported that ZBF is compatible with analysis of cell surface immunophenotype and detection of intracellular epitopes by flow cytometry. The aim of this study was to explore whether ZBF is also compatible with the detection of DNA damage response assessed by phospho-specific antibodies (Abs) detecting phosphorylation of the key proteins of that pathway. DNA damage in human pulmonary adenocarcinoma A549 cells was induced by treatment with the DNA topoisomerase I inhibitor camptothecin and phosphorylation of histone H2AX on Ser139 (γH2AX) and of ATM on Ser1981 was detected with phospho-specific Abs; cellular fluorescence was measured by laser scanning cytometry (LSC). The sensitivity and accuracy of detection of H2AX and ATM phosphorylation concurrent with the detection of DNA replication by EdU incorporation and "click chemistry" was found in ZBF fixed cells to be comparable to that of cell fixed in formaldehyde. The accuracy of DNA content measurement as evident from the resolution of DNA content frequency histograms of cells stained with DAPI was somewhat better in ZBF- than in formaldehyde-fixed cells. The pattern of chromatin condensation revealed by the intensity of maximal pixel of DAPI that allows one to identify mitotic and immediately post-mitotic cells by LSC was preserved after ZBF fixation. ZBF fixation was also compatible with the detection of γH2AX foci considered to be the hallmarks of induction of DNA double-strand breaks. Analysis of cells by flow cytometry revealed that ZBF fixation of lymphoblastoid TK6 cells led to about 60 and 33% higher intensity of the side and forward light scatter, respectively, compared to formaldehyde fixed cells.

The role of chromatin condensation during granulopoiesis in the regulation of gene cluster expression

Changes in nuclear architecture play an important role in the regulation of gene expression. The importance of epigenetic changes is observed during granulopoiesis, when changes in the nuclear architecture are considered a major factor that influences the downregulation of genes. We aimed to assess the influence of chromatin condensation on the regulation of gene expression during granulopoiesis. Based on a previously published microarray analysis, we chose loci with different levels of transcriptional activity during granulopoiesis. Fluorescent in situ hybridisation (FISH) and immunofluorescent labelling of RNA polymerase II were used to determine the relationship between the transcriptional activity of gene clusters and their localisation within areas with different levels of chromatin condensation. Although active loci were positioned outside of areas of condensed chromatin, downregulation of genes during granulopoiesis was not accompanied by a shift of the downregulated loci to condensed areas. Only the beta-globin cluster was subjected to chromatin condensation and localised to condensed areas. Our results indicate that granulopoiesis is accompanied by a non-random, tissue-specific pattern of chromatin condensation. Furthermore, we observed that the decrease in the quantity of RNA polymerase II correlates with the differentiation process and likely acts in synergy with chromatin condensation to downregulate total gene expression.

Spermatic cell characteristics in Gymnotiformes (Teleostei: Ostariophysi) and their phylogenetic meaning

Spermatic characteristics were studied in representatives of the families Rhamphichthyidae, Sternopygidae and Apteronotidae, and compared with pre-existent data from Gymnotidae and Hypopomidae. The spermatic characteristics found in Gymnotiformes were also compared with data from other Ostariophysi spermatic cells. The spermatic characteristics as the type of spermiogenesis and the structural pattern of the sperm, considering nuclear form, pattern of chromatin condensation, nuclear fossa and its relation with the centriolar complex, form and localization of mitochondria, form of midpiece, presence or absence of cytoplasmic canal and cytoplasmic sleeve, and flagellar fins were utilized. The comparative analysis of the spermatic cell of Gymnotiformes better support the previous proposals for the order considering Gymnotidae as a derived group, than the more recent ones that located Gymnotidae in a basal position as the sister group of the remaining Gymnotiformes. Regarding the Ostariophysi, the comparative analyses based on the spermatic cell characteristics is consistent with a recent systematic proposal that consider Gymnotiformes as a sister group of Characiformes.

Entropy of Feulgen-stained 17-β-estradiol-transformed human breast epithelial cells as assessed by restriction enzymes and image analysis

MCF-10F human breast epithelial cells when transformed with 17-beta-estradiol (E2) give rise to highly invasive C5 cells that generate adenocarcinomas in SCID mice. From these tumors, cell lines such as C5-A6-T6 and C5-A8-T8 have been derived. Variable patterns of chromatin supraorganization have been demonstrated for these cells during the transformation/tumorigenesis progress, when assessing chromatin entropy by image analysis in Feulgen-stained preparations. Since epigenetic dysregulation might contribute to the chromatin textural repatterning in transformed MCF-10F cells, the association of the variable chromatin packing states with global DNA methylation was investigated in these cells after their treatment with restriction enzymes followed by Feulgen staining and chromatin entropy evaluation by image analysis. The results indicate that although -CmCGG- sequences may affect chromatin supraorganization in some of the analyzed cell types (perhaps due to localized hypermethylation), not all the chromatin condensation patterns in these cells with transformation and/or tumorigenesis are associated with DNA methylation (e.g. E2 cells). Chromatin supraorganization remodeling in C5-A6-T6 and C5-A8-T8 cells may be attained by different mechanisms, with C5-A6-T6 chromatin packing states perhaps being associated with local DNA hypermethylation or other epigenetic factors, and C5-A8-T8 likely being associated with global DNA hypomethylation, as reported in the literature for other cell types. Thus, we assume that a variable epigenetic modulation affecting the higher-order packing states of chromatin in the estrogen-transformed MCF-10F cell model could be evident with the chromatin entropy study by image analysis.