Diffuse Chromatin Research Articles

DNA Damage in Bat Blood Leukocytes Using a Chromatin Dispersion Test (CDT): Biomarker of Environmental Genotoxicity.

Environmental pollutants produce adverse effects on organisms and ecosystems. Biomonitoring and biomarkers offer a reasonable approach to make these assessments. Induced genetic changes can be using as a biomarker in organisms that react to a given compound in the ecosystem. Monitoring environmental genotoxicity necessitates the choice of model animals known as "sentinels or biological monitors" and the suitability of validated tests for DNA damage evaluation. We aimed to estimate the DNA damage produced by thermal stress in the leukocytes of the Mexican free-tailed bat (Tadarida brasiliensis). The DNA damage in bat leukocytes exposed to different temperatures (35°C, 45°C, and 55°C) was evaluated by the adapted chromatin dispersion test (CDT) and the results were confirmed by the alkaline comet test. The CDT permitted a clear representation of leukocytes with fragmented DNA and of nonfragmented DNA. In addition, we detected nuclear anomalies in relation to cell death cellular swelling, nuclear fragmentation, and chromatin lysis. The alkaline comet assay revealed that the halos of diffuse chromatin include fragmented DNA. The assay of the method employing the CDT is well established, precise, and cost-effective for the routine quantitative analysis of DNA damage on the effect of the leukocytes of bats exposed to thermal stress. This could also apply as a sensitive screening tool for the evaluation of genotoxicity in environmental protection programs.

Transcriptionally active chromatin loops contain both ‘active’ and ‘inactive’ histone modifications that exhibit exclusivity at the level of nucleosome clusters

Chromatin state is thought to impart regulatory function to the underlying DNA sequence. This can be established through histone modifications and chromatin organisation, but exactly how these factors relate to one another to regulate gene expression is unclear. In this study, we have used super-resolution microscopy to image the Y loops of Drosophila melanogaster primary spermatocytes, which are enormous transcriptionally active chromatin fibres, each representing single transcription units that are individually resolvable in the nuclear interior. We previously found that the Y loops consist of regular clusters of nucleosomes, with an estimated median of 54 nucleosomes per cluster with wide variation.In this study, we report that the histone modifications H3K4me3, H3K27me3, and H3K36me3 are also clustered along the Y loops, with H3K4me3 more associated with diffuse chromatin compared to H3K27me3. These histone modifications form domains that can be stretches of Y loop chromatin micrometres long, or can be in short alternating domains. The different histone modifications are associated with different sizes of chromatin clusters and unique morphologies. Strikingly, a single chromatin cluster almost always only contains only one type of the histone modifications that were labelled, suggesting exclusivity, and therefore regulation at the level of individual chromatin clusters. The active mark H3K36me3 is more associated with actively elongating RNA polymerase II than H3K27me3, with polymerase often appearing on what are assumed to be looping regions on the periphery of chromatin clusters.These results provide a foundation for understanding the relationship between chromatin state, chromatin organisation, and transcription regulation – with potential implications for pause-release dynamics, splicing complex organisation and chromatin dynamics during polymerase progression along a gene.

Dissecting the role of H3K27 acetylation and methylation in PRC2 mediated control of cellular identity

The Polycomb repressive complexes PRC1 and PRC2 act non-redundantly at target genes to maintain transcriptional programs and ensure cellular identity. PRC2 methylates lysine 27 on histone H3 (H3K27me), while PRC1 mono-ubiquitinates histone H2A at lysine 119 (H2Aub1). Here we present engineered mouse embryonic stem cells (ESCs) targeting the PRC2 subunits EZH1 and EZH2 to discriminate between contributions of distinct H3K27 methylation states and the presence of PRC2/1 at chromatin. We generate catalytically inactive EZH2 mutant ESCs, demonstrating that H3K27 methylation, but not recruitment to the chromatin, is essential for proper ESC differentiation. We further show that EZH1 activity is sufficient to maintain repression of Polycomb targets by depositing H3K27me2/3 and preserving PRC1 recruitment. This occurs in the presence of altered H3K27me1 deposition at actively transcribed genes and by a diffused hyperacetylation of chromatin that compromises ESC developmental potential. Overall, this work provides insights for the contribution of diffuse chromatin invasion by acetyltransferases in PRC2-dependent loss of developmental control.

Morphological and ultrastructural analysis of normal, injured and osteoarthritic human knee menisci.

The human meniscus plays a crucial role for transmission and distribution of load across the knee, as well as shock absorption, joint stability, lubrication, and congruity. The aim of this study was to compare the complex geometry, and unique ultrastructure and tissue composition of the meniscus in healthy (control) and pathological conditions to provide understanding of structural changes that could be helpful in the future design of targetted therapies and improvement of treatment indications. We analyzed meniscus samples collected from 3 healthy multi-organ donors (median age, 66 years), 5 patients with traumatic meniscal tear (median age, 41 years) and 3 patients undergoing total knee replacement (TKR) for end-stage osteoarthritis (OA) (median age, 72 years). We evaluated the extracellular matrix (ECM) organization, the appearance and distribution of areas of calcification, and modifications of cellular organization and structure by electron microscopy and histology. The ECM structure was similar in specimens from traumatic meniscus tears compared to those from patients with late-stage OA, showing disorganization of collagen fibers and increased proteoglycan content. Cells of healthy menisci showed mainly diffuse chromatin and well preserved organelles. Both in traumatic and in OA menisci, we observed increased chromatin condensation, organelle degeneration, and cytoplasmic vacuolization, a portion of which contained markers of autophagic vacuoles. Areas of calcification were also observed in both traumatic and OA menisci, as well as apoptotic- like features that were particularly prominent in traumatic meniscal tear samples. We conclude that meniscal tissue from patients with traumatic meniscal injury demonstrate pathological alterations characteristic of tissue from older patients undergoing TKR, suggesting that they have high susceptibility to develop OA.

Abstract 3167: Loss of HP1α alters the integrity of the nuclear lamina, thereby promoting invasion

Abstract Malignant cell invasion is accompanied by complex changes in nuclear morphology and chromatin patterning that have been utilized by pathologists to determine malignancy. Members of the HP1 family modulate chromatin plasticity, with the HP1α paralog required to maintain the condensation of transcriptionally silent heterochromatin and to sequester it at the nuclear periphery. While increased expression of HP1α is proposed to aid the onset of malignancy, the acquisition of an invasive phenotype correlates with the loss of this architectural protein in many solid tumors including those of the thyroid, kidney, colon and breast. We have shown in a Drosophila model of epithelial cell invasion that loss of HP1α synergistically enhances invasion in cells with an activated oncogenic pathway. In the poorly invasive MCF7 breast cells, loss of HP1α increases their invasive potential and promotes epithelial plasticity. These phenotypic changes are associated altered nuclear morphology including increased nuclear grooves and indentations, diffuse chromatin patterning and a loss of heterochromatin at the nuclear periphery. Since during metastatic invasion, the nucleus, the largest and most rigid cellular of organelle, becomes malleable enough to squeeze through interstitial spaces in the surrounding tissue. This lead us to propose that loss of HP1α causes a remodelling of nuclear periphery thereby increasing nuclear malleability. Here we demonstrate that HP1α knockdown in MCF7 cells decreases mechanical stability of the nuclei and altering the integrity of nuclear lamina. While total levels of Lamin A/C, Lamin B1 and Lamin B2 are unchanged within the cell, their solubility and thus localization within the nucleus is altered. We also demonstrate how this change in the lamina effects other proteins that function at the nuclear periphery. Together, these findings suggest that when loss of HP1α increases invasive potential there is a corresponding disruption to the nuclear periphery, creating a weakness in these cells that could be exploited for therapeutic benefit. Citation Format: Tracy K. Hale, Raoul Solomon. Loss of HP1α alters the integrity of the nuclear lamina, thereby promoting invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3167.

169 Nuclear Maturation Kinetics and In Vitro Fecundation of Immature Bovine Oocytes Injected into Preovulatory Follicles

The objective was to evaluate in vitro nuclear maturation and fecundation kinetics of oocytes injected into preovulatory follicles of synchronized cows using the intra-follicular oocyte injection (IFOI) technique. In experiment 1, 438 immature abattoir-bovine cumulus–oocyte complexes (COC) of grades I, II, and III were randomly allocated to 1 of 3 groups: Matvitro (n = 111), COC matured in vitro for 22 h; Matvivo20 (n = 172) and Matvivo30 (n = 155), 30 oocytes were injected into each preovulatory follicle of pre-synchronized recipients. In Matvivo20, oocytes were matured for 19.8 ± 0.1 h and in Matvivo30, for 28.3 ± 0.1 h. All cows received 12.5 mg of LH (Lutropin, Bioniche, Canada) at IFOI (Matvivo20) or 10 h after IFOI (Matvivo30). Oocytes from Matvivo20 and Matvivo30 were aspirated 20 h after LH injection for assessment of oocyte maturation and recovery rates. Oocytes were evaluated according to maturation kinetics as germinal vesicle, metaphase I, anaphase I, telophase I, metaphase II, parthenogenetically activated, and degenerated (chromosomal aberrations, presence of diffuse or indefinite chromatin). In experiment 2, immature abattoir-bovine COC (n = 202) of grades I, II, and III were randomly distributed into 2 groups: Matvitro (n = 103), COC were matured and fertilized in vitro; Matvivo (n = 99), same as Matvivo20 protocol, and COC fertilized in vitro. Presumptive zygotes were evaluated as fertilized, unfertilized, or polyspermic. Statistical analyses were performed by the GLIMMIX procedure of SAS (SAS Institute Inc., Cary, NC, USA). Recovery rate was lower (P < 0.001) in Matvivo20 (52.9%, 91/172) compared with Matvivo30 (72.9%, 113/155). Germinal vesicle (P = 0.94), metaphase I (P = 0.98), anaphase I (P = 0.99), and telophase I (P = 0.20) rates were similar. However, there were differences in metaphase II [Matvitro: 81.0% (90/111)a, Matvivo20: 74.5% (35/47)a, and Matvivo30: 41.6% (32/77)b; P = 0.001], degenerate [Matvitro: 5.4% (6/111)c, Matvivo20: 21.3% (10/47)b and Matvivo30: 48.1% (37/77); P = 0.001] and parthenogenetically activated [Matvitro: 0.0% (0/111)b, Matvivo20: 0.0% (0/47)b and Matvivo30: 9.1% (7/77)a; P = 0.001]. Polyspermic (P = 0.18) and abnormal (P = 0.98) rates were similar. However, there was a higher rate (P = 0.05) of fertilized oocytes in Matvivo (60.6%, 60/99) than in Matvitro (46.6%, 48/103). In conclusion, oocyte maturation in vivo after IFOI for 20 h does not alter maturation kinetics and increases in vitro oocyte fertilization capacity. However, the 10-h increase in intra-follicular oocyte permanence decreased the proportion of viable oocytes. Thus, the oocyte maturation phase is not the limiting causative factor for the low IFOI-embryo production rates.

CYTOMORPHOLOGICAL RESEARCH OTSYT-CUMULUS COMPLEXES RABBIT FROM WITH OVARIAN AT DIFFERENT STAGES OF THE ESTROUS CYCLE

The analysis of the research results revealed that the largest number (86.4%) of oocytes suitable for further development outside the body can be obtained with ovarian follicular phase of growth. It should be noted that statistically significant difference was observed between the groups OCC rabbit derived from ovaries at different phases of the estrous cycle by the number oocytes unsuitable for further cultivation. Thus, the phase of the ovarian follicular growth of gametes was obtained only 13.6% of ovarian and with signs of ovulation and the luteal phase, 35.4% and 31.4% respectively. When comparing the results of the analysis of cytogenetic preparations oocytes from ovaries removed rabbit at different stages of the estrous cycle, found that regardless of the phase of the estrous cycle Yachnik mostly larger number of oocytes were under dyploteny. The largest number of gametes with diffuse chromatin at the stage dyploteny (37.3%) received from the stage ovarian follicular growth. At the stage of fibrillar dyploteny increasing number of gametes was removed from ovarian luteal phase of the estrous cycle. In step dyploteny visible bivalent were more likely gametes obtained from stage ovarian follicular growth (18,1%, p <0,05). The highest percentage of oocytes degeneration chromatin was observed in the group removed from the ovaries to the rabbit lyutealniy phase (21.6%).

Three-dimensional apoptotic nuclear behavior analyzed by means of Field Emission in Lens Scanning Electron Microscope.

Apoptosis is an essential biological function required during embryogenesis, tissue home-ostasis, organ development and immune system regulation. It is an active cell death pathway involved in a variety of pathological conditions. During this process cytoskeletal proteins appear damaged and undergo an enzymatic disassembling, leading to formation of apoptotic features. This study was designed to examine the three-dimensional chromatin behavior and cytoskeleton involvement, in particular actin re-modeling. HL-60 cells, exposed to hyperthermia, a known apoptotic trigger, were examined by means of a Field Emission in Lens Scanning Electron Microscope (FEISEM). Ultrastructural observations revealed in treated cells the presence of apoptotic patterns after hyperthermia trigger. In particular, three-dimensional apoptotic chromatin rearrangements appeared involving the translocation of filamentous actin from cytoplasm to the nucleus. FEISEM immunogold techniques showed actin labeling and its precise three-dimensional localization in the diffuse chromatin, well separated from the condensed one. The actin presence in dispersed chromatin inside the apoptotic nucleus can be considered an important feature, indispensable to permit the apoptotic machinery evolution.

Chromosome Condensation: Weaving an Untangled Web

The compaction of diffuse interphase chromatin into stable mitotic chromosomes enables the segregation of replicated DNA to daughter cells. Two new studies characterise, both invivo and invitro, the essential contribution of the vertebrate condensin complex to chromosome organisation.

Anti-Apoptotic Effects of Platelet Factor 4 on Human T-Lymphocytes

In addition to a major role in hemostasis and thrombosis, platelets have many other functions, including modulation of the immune response. Activated platelets release a number of potent chemokines, such as NAP-2 (CXCL7), RANTES (CCL5), and platelet factor 4 (PF4; CXCL4). PF4 is stored in platelet α-granules at very high concentrations and is released upon cell activation at the sites of inflammation and injury. PF4 is known as a key player in heparin-induced thrombocytopenia, a severe autoimmune complication of heparin therapy, but the roles of PF4 in other immunogenic pathologies are not well understood. In this study, we evaluated the direct effects of PF4 on human T-lymphocytes in vitro with respect to apoptosis.T-cells were immunomagnetically isolated from the blood of healthy donors and cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS, antibiotics (penicillin and streptomycin) and L-glutamine during 24 hours in the absence or presence of recombinant wild-type human PF4. Aliquots were taken after 2, 4, 6, 12 and 24 hours of cultivation and the cells were analyzed for ultrastructural and biochemical signs of apoptosis using transmission electron microscopy, flow cytometry, and Western blot analysis.The vast majority of control T-lymphocytes had a morphology corresponding to time-dependent changes characteristic of different stages of apoptosis. At the early stages (up to 6 hours) the cells lost microvilli from the plasma membrane. At the later stages deep invagination of nuclear and plasma membranes as well as chromatin condensation were observed. The plasma membrane formed blebs and a lot of fragments of dead cells (apoptotic bodies) were revealed. The mitochondria, endoplasmic reticulum and Golgi apparatus were clearly visible in the cytoplasm.The addition of PF4 to the cells at a final concentration of 2 μg/ml reduced the degree of apoptotic changes. Many T-cells still developed the morphological changes associated with apoptotic cells; however most of them did not progress past the described early stages of apoptosis, even up to 24 hours. These cells maintained a regular oval shape, but the nuclear membrane formed invaginations and chromatin condensed and relocated to the nuclear periphery. The apoptotic bodies were less frequent than in control. The mitochondria, endoplasmic reticulum, vacuoles and lysosomes were well distinguished in the cytoplasm but their number was decreased. However, there was also a subpopulation of T-cells that had an oval shape, the nucleus and plasma membrane were without invaginations, the nucleus was roundish with diffuse chromatin and the nucleolus was clearly seen in the nucleus. The morphology of this subpopulation corresponds to proliferated cells.In the presence of PF4 added at a final concentration of 100 μg/ml most of the cells kept the typical morphology of proliferating cells up to 24 hours. The plasma membrane had a regular round shape, it was smooth and without invaginations. The nucleus usually occupied a large part of the cell; it was rounded and located centrally. The nucleolus was clearly visualized in the nucleus and the nuclear substance was distributed homogeneously. The ribosomes, the Golgi apparatus and the endoplasmic reticulum were well distinguished in the cytoplasm. The mitochondria had a regular shape with well-differentiated cristae. Most often, accumulation of the mitochondria was observed medially, near the nucleus.The observed morphological changes were correlated with expression of molecular markers and other signs of apoptosis. Bcl-2 and caspase-3 were not expressed in the T-cells treated with PF4 at a concentration of 100 µg/ml, while caspase-3 was detected in the control cells and in the cells treated with PF4 at a concentration of 2 µg/ml after 4 and 6 hours of incubation. Consistent results were obtained with assessment of the degree of apoptosis by Annexin V binding and propidium iodide staining.In conclusion, our results provide direct evidence for dose-dependent anti-apoptotic effects of PF4 on T-cells, suggesting that PF4 may sustain an immune response by extending T-lymphocyte survival.(Research supported by NIH grant HL110860 and the Program of Competitive Growth of Kazan Federal University) DisclosuresNo relevant conflicts of interest to declare.

Intracellular amyloid and the neuronal origin of Alzheimer neuritic plaques

Genetic analysis of familial forms of Alzheimer's disease (AD) causally links the proteolytic processing of the amyloid precursor protein (APP) and AD. However, the specific type of amyloid and mechanisms of amyloid pathogenesis remain unclear. We conducted a detailed analysis of intracellular amyloid with an aggregation specific conformation dependent monoclonal antibody, M78, raised against fibrillar Aß42. M78 immunoreactivity colocalizes with Aß and the carboxyl terminus of APP (APP-CTF) immunoreactivities in perinuclear compartments at intermediate times in 10month 3XTg-AD mice, indicating that this represents misfolded and aggregated protein rather than normally folded APP. At 12months, M78 immunoreactivity also accumulates in the nucleus. Neuritic plaques at 12months display the same spatial organization of centrally colocalized M78, diffuse chromatin and neuronal nuclear NeuN staining surrounded by peripheral M78 and APP-CTF immunoreactivity as observed in neurons, indicating that neuritic plaques arise from degenerating neurons with intracellular amyloid immunoreactivity. The same staining pattern was observed in neuritic plaques in human AD brains, showing elevated intracellular M78 immunoreactivity at intermediate stages of amyloid pathology (Braak A and B) compared to no amyloid pathology and late stage amyloid pathology (Braak 0 and C, respectively). These results indicate that intraneuronal protein aggregation and amyloid accumulation is an early event in AD and that neuritic plaques are initiated by the degeneration and death of neurons by a mechanism that may be related to the formation of extracellular traps by neutrophils.

Cytochemical study of the embryo sac of Nicotiana tabacum L.

The mitotic cycle in the egg cell and physico-chemical state of chromatin in the egg cell and central cell of the tobacco embryo sac were studied. It was revealed that during egg cell formation a change in the mitotic cycle kinetics takes place, it consists in prolongation of the S-period as compared with that of somatic cells and G1 - period as compared with that of male gametes. Egg cell and central cell nuclei differ in chromatin structure. Condensed chromatin dominates in the egg cell nucleus, diffuse chromatin in the central cell nucleus, but both show only weak metabolic activity.

Ultrastructural study of spermiogenesis in a rare desert amphisbaenian Diplometopon zarudnyi

Spermiogenesis, in particular the head differentiation of Diplometopon zarudnyi, was studied at the ultrastructural level by Transmission Electron Microscope (TEM). The process includes acrosomal vesicle development, nuclear elongation, chromatin condensation and exclusion of excess cytoplasm. In stage I, the proacrosomal vesicle occurs next to a shallow fossa of the nucleus, and a dense acrosomal granule forms beneath it. This step commences with an acrosome vesicle forming from Golgi transport vesicles; simultaneously, the nucleus begins to move eccentrically. In stage II, the round proacrosomal vesicle is flattened by projection of the nuclear fossa, and the dense acrosomal granule diffuses into the vesicle as the fibrous layer forms the subacrosomal cone. Circular manchettes surrounded by mitochondria develop around the nucleus, and the chromatin coagulates into small granules. The movement of the nucleus causes rearrangement of the cytoplasm. The nucleus has uniform diffuse chromatin with small indices of heterochromatin. The subacrosome space develops early, enlarges during elongation, and accumulates a thick layer of dark staining granules. In stage III, the front of the elongating nucleus protrudes out of the spermatid and is covered by the flat acrosome; coarse granules replace the small ones within the nucleus. One endonuclear canal is present where the perforatorium resides. In stage IV, the chromatin concentrates to dense homogeneous phase. The circular manchette is reorganized longitudinally. The Sertoli process covers the acrosome and the residues of the cytoplasmic lobes are removed. In stage V, the sperm head matures.

ULTRASTRUCTURAL STUDY OF BALANTIDIUM POLYVACUOLUM LI, 1963 (CLASS: LITOSTOMATEA) THAT INHABITS IN XENOCYPRINAE FISH

Balantidium polyvacuolum Li,1963,an obligatory intestinal symbiont of Xenocyprinae fish,mainly inhabits in the luminal contents of the middle and hindgut.Though the description and some revisions are given at both light and scanning electronic microscopic levels,the morphological data are still incomplete since fine structural data with taxonomic values are still unavailable.This study examined the ultrastructural anatomy of this Chinese balantidium that inhabits in the intestines of Xenocypris davidi collected from the Jialing River in Sichuan province during fish parasite surveys in August 2011.All fish samples were dissected,and intestines were cut to collect the luminal contents into petridishes for further examination with the aid of a dissecting microscope.The luminal contents of the hindgut containing numerous B.polyvacuolum were selected and fixed directly in 2.5% glutaraldehyde in 0.2 M phosphate buffered saline(PBS,PH 7.4) for 2h at 4 ℃,then postfixed in 1%(v/v) osmium tetroxide in PBS for 2h at 4 ℃,this step was followed by a series of dehydration in acetone and embedded in Araldite.Ultrathin sections were cut on a Leica Ultracut R ultramicrotome,stained with uranyl acetate and lead citrate before being observed in a Philips Tecnai G2 20 Transmission Electron Microscope(Netherlands).Its somatic cortex,vestibulum,nuclei,cytoplasmic organelles and the centroventral concave platelet were well described here.Our results indicated that both somatic cortex and vestibulum were composed of pellicle(the outermost cell membranes,pellicular alveoli and the layer of microtubules) and infraciliature(kinetosomes and associated fibrils such as kinetodesmal fibril,postciliary microtubules,transverse microtubules and nematodesmata).However,the nematodesmata of vestibular kinetids were much longer than those of somatic kinetids and usually extended deeply into the endoplasm.As for the nuclei,the macronucleus was club-shaped with several nucleoli and heterochromatin occupying the karyoplasm,whereas the spherical micronucleus was homoeomerous with diffuse chromatin distributed over the karyoplasm.In addition,large amounts of amylopectin granules were observed within cytoplasm of the centroventral concave platelet.We suspected that this region may act as a storage pool of amylose to provide energy for the organism.Besides,the components of the cortex were compared among the related Balantidium species and their possible functions were also discussed.

Chromatin Patterns of Immature Canine Oocytes after In Vitro Maturation

In canine species, in vitro maturation (IVM) rates of oocytes collected from anoestrous ovaries are low (<20%). Several IVM media have been tested without significant improvements. A critical step in the evaluation of culture conditions is the observation of the meiotic stage reached by the oocytes. The present study was designed to investigate the chromatin patterns of in vitro matured oocytes by visualizing Germinal Vesicle (GV) and Germinal Vesicle Breakdown (GVBD) structures at 72 h of IVM. Nuclear stages of 1678 oocytes were evaluated by confocal microscopy after IVM. 1204 oocytes were non-degenerated, and 94.4% were still immature and at GV stage. Five different patterns of chromatin configuration were observed. Higher percentages of oocytes with unmodified GV and with diffuse (58%; Type A) and filamentous chromatin (19%; Type B) were observed in comparison with those with modifications in the GV such as patched chromatin (12.5%; Type C), surrounded-nucleolus (3%; Type D) and in vivo type chromatin/fully grouped chromatin (2.5%; Type E). These results indicate that GVBD (absence of nucleolus, nucleus breakdown) is rarely observed in vitro. The percentage of type C-D-E GVs and MI (meiotic resumption) and of MII (completion of meiosis) can be used to evaluate meiotic resumption after IVM. Our results indicate that although a low number of in vitro matured oocytes exhibit the chromatin configurations observed in in vivo collected oocytes, chromatin changes in the GV can be induced during IVM.

Ultrastructure of spermiogenesis in the American alligator,Alligator mississippiensis(Reptilia, Crocodylia, Alligatoridae)

Testicular samples were collected to describe the ultrastructure of spermiogenisis in Alligator mississipiensis (American Alligator). Spermiogenesis commences with an acrosome vesicle forming from Golgi transport vesicles. An acrosome granule forms during vesicle contact with the nucleus, and remains posterior until mid to late elongation when it diffuses uniformly throughout the acrosomal lumen. The nucleus has uniform diffuse chromatin with small indices of heterochromatin, and the condensation of DNA is granular. The subacrosome space develops early, enlarges during elongation, and accumulates a thick layer of dark staining granules. Once the acrosome has completed its development, the nucleus of the early elongating spermatid becomes associated with the cell membrane flattening the acrosome vesicle on the apical surface of the nucleus, which aids in the migration of the acrosomal shoulders laterally. One endonuclear canal is present where the perforatorium resides. A prominent longitudinal manchette is associated with the nuclei of late elongating spermatids, and less numerous circular microtubules are observed close to the acrosome complex. The microtubule doublets of the midpiece axoneme are surrounded by a layer of dense staining granular material. The mitochondria of the midpiece abut the proximal centriole resulting in a very short neck region, and possess tubular cristae internally and concentric layers of cristae superficially. A fibrous sheath surrounds only the axoneme of the principal piece. Characters not previously described during spermiogenesis in any other amniote are observed and include (1) an endoplasmic reticulum cap during early acrosome development, (2) a concentric ring of endoplasmic reticulum around the nucleus of early to middle elongating spermatids, (3) a band of endoplasmic reticulum around the acrosome complex of late developing elongate spermatids, and (4) midpiece mitochondria that have both tubular and concentric layers of cristae.

Spermiogenesis particularities of a sperm storage species: &lt;i&gt;Helicolenus dactylopterus&lt;/i&gt; (Teleostei: Scorpaenidae)

SUMMARY: A study of the spermiogenesis and spermatozoa of Helicolenus dactylopterus was conducted. Females of this species have the capacity to store sperm within their ovaries, and male gametes have a considerable cytoplasmic mass surrounding their heads to survive the long period of intraovarian sperm storage. Our observations show that early spermatids are round-shaped cells and have a spherical nucleus with diffuse chromatin. The nuclear volume decreases as a result of progressive chromatin condensation during spermiogenesis, causing the nucleus to take on a U-shape. Flagellar insertion is not central to the nucleus but consistently occurs at an oblique angle towards one side of it. The flagellum is inserted into the nuclear fossa, without subsequent nuclear rotation. In mature spermatozoa, the flagellum is adjacent to the nucleus. A comparison of the spermatozoa in the testicular lobules and those in the intraovarian storage structures suggests that the increase in volume of the cytoplasmic mass may occur in the posterior region of the testis, in the testicular duct. Spermatozoa enter the ovary in groups that reach the ovarian lumen and are surrounded by the ovarian epithelium for storage in sperm storage crypts.

Apoptotic DNA fragmentation can be revealed in situ: An ultrastructural approach

A common pattern of apoptotic death is DNA cleavage, initially producing large fragments (50 kbp), followed by the production of nucleosomic/oligonucleosomic fragments. Nevertheless, apoptosis without DNA fragmentation, at least of the nucleosomic type, has been reported. To investigate the spatial relationship between DNA cleavage and chromatin condensation, we applied the TUNEL technique to the ultrastructural analysis of apoptotic cells. A modified method, utilizing a gold-conjugated antidigoxigenin antibody, was carried out on U937 versus Molt-4 cells, both exposed to UVB radiation or staurosporine treatment. Gold particle density in the different domains of apoptotic cells was evaluated by a four-way ANOVA test. Gold labelling was more strongly localised in condensed chromatin than in the diffuse chromatin. U937 cells, which evidenced in vitro oligonucleosomic fragmentation after both UVB and staurosporine treatments, revealed a significantly higher gold particle density, when compared with Molt-4, which did not show, on the other hand, oligonucleosomic cleavage even in the presence of < or = 50 kbp cleavage. Thus, a correlation between DNA fragment sizes and gold particle density appears. TUNEL applied to electron microscopy is an effective approach to study the relationship between apoptotic chromatin condensation and DNA cleavage. Both these events indeed appear in the apoptotic nucleus, but their reciprocal correlation is still greatly unknown. Microsc. Res. Tech. 2009. (c) 2009 Wiley-Liss, Inc.

Gross Cystic Disease Fluid Protein—(GCDFP-15)

A total of 211 cases of primary lung adenocarcinoma were tested for expression of gross cystic disease fluid protein-15 (GCDFP-15) and only 11 cases (5.2%) were positive. The cases occurred with an equal sex distribution in older individuals whose carcinomas were frequently identified on screening radiographs. The adenocarcinomas were peripheral lesions and had an average size of 2.9 cm (range, 1.1 to 7.0). Histologically, they were usually mixed acinar and papillary adenocarcinomas with abundant extracellular mucin production, with the neoplastic cells having a polygonal shape, round to oval nuclei, diffuse powdery chromatin, and abundant eosinophilic granular cytoplasm. Clear cells and apocrinelike cells with prominent central nucleoli were common. GCDFP-15 was expressed in conjunction with thyroid transcription factor-1 in 81% of cases and synaptophysin was seen in 65%. Estrogen and progesterone receptors were not expressed. EGFR gene amplification and mutations of exons 19 and 21 were rare. KRAS mutations and HER2 gene amplification were not seen. This report details the first 11 cases of pulmonary adenocarcinoma to express GCDFP-15 and their distinctive morphology with frequent mucin production and coexpression of thyroid transcription factor-1 and synaptophysin.

There's more than one way to skin a cat: Yet another way to assess mitotic index by cytometry

There's more than one way to skin a cat: Yet another way to assess mitotic index by cytometry