Pattern Of Chromatin Condensation Research Articles

Non-genotoxic hepatocarcinogenesis in vitro: the FaO hepatoma line responds to peroxisome proliferators and retains the ability to undergo apoptosis

A range of hepatoma cell lines (RH1, HTC, FaO, 7800C1 and MH1C1), has been studied with the aim of establishing an in vitro model to investigate the molecular mechanisms of hepatocarcinogenicity induced by the peroxisome proliferator class of non-genotoxic carcinogens. In view of speculation that peroxisome proliferators suppress hepatocyte apoptosis in vivo, we have placed particular emphasis on evaluating whether hepatoma cell lines retain the ability to undergo apoptotic cell death. Expression of the liver-specific differentiation marker albumin and the peroxisome proliferator-activated receptor (PPAR) was highest in the Reuber hepatoma cell line, FaO. This cell line also demonstrated the most marked response to the peroxisome proliferator nafenopin with a 2.2-fold induction of the microsomal enzyme cytochrome p450IVA1. This response was found to display intercellular heterogeneity by immunocytochemistry. Thus, the FaO cell line maintained characteristics of hepatocytes, both in vivo and in vitro, in terms of expression of constitutive and inducible markers. However, none of the cell lines tested mirrored the hyperplastic response of hepatocytes to nafenopin, since no increase in cell growth kinetics was observed on addition of nafenopin to the growth medium. The mode of cell death in confluent FaO cultures was characterised as apoptosis, by fluorescence microscopy and agarose gel electrophoresis of extracted DNA. Cells detaching from confluent FaO cultures exhibited chromatin condensation and DNA fragmentation patterns characteristic of cels undergoing apoptotic death.Interestingly, no apoptosis was seen in monolayer cells, suggesting that apoptosis in vitro is associated with cell shrinkage and detachment similar to that documented for the liver in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of DNase I-hypersensitive regions during rat spermatogenesis: stage-dependent patterns and unique sensitivity of elongating spermatids.

DNase I-hypersensitivity of rat spermatogenic cells was analyzed 1) to establish overall patterns of hypersensitivity in individual cell types, 2) to correlate these patterns with known changes in chromatin organization and function, and 3) to provide a foundation for further analyses examining DNase I-hypersensitivity and the localization of specific genes during spermatogenesis. Parameters for in situ nick translation, using radioactive and fluorescent probes to visualize DNase I-hypersensitive regions (DHR), were established for fixed and sectioned testicular preparations, permeabilized cells, and isolated germ cell nuclei. As anticipated, the pattern of DHR changed in a cell-type specific manner during the course of spermatogenesis, reflective of known stage-dependent alterations in the composition and structure of both the chromatin and the nuclear lamina/matrix as well as changes in gene expression. DHR in preleptotene spermatocytes were primarily peripheral, while in pachytene spermatocytes they were localized along the condensed chromosomes. The pattern of DHR changed from "checkerboard" in steps 7-8 round spermatid nuclei to "lamellar" in steps 10-11 elongating spermatids. In steps 12-13 elongating spermatids. DHR were localized throughout the nuclei or in a graded manner--increasing from anterior to posterior and mirroring the pattern of chromatin condensation. However, unlike the case in other stages, DNA of steps 12-13 elongating spermatids was exquisitely sensitive to nick translation even in the absence of exogenous DNase I. In contrast to the labeling of earlier stages, steps 16-19 spermatids and mature spermatozoa did not demonstrate DNase I-hypersensitivity under any conditions employed. A variety of agents that interact with topoisomerase II and DNA (teniposide, novobiocin, ethidium bromide, and adenosine triphosphate) were tested to determine the basis for the unique sensitivity to nick translation of steps 12-13 elongating spermatids. None of the agents tested, however, affected this unique labeling. The sensitivity of steps 12-13 elongating spermatids to nick translation in the absence of exogenous nuclease indicators the presence of endogenous nicks, which may relieve torsional stress and aid rearrangement as the chromatin is packaged into a form characteristic of the mature spermatozoon.

Ultrastructural changes in hypothalamic supraoptic nucleus neurons of ovariectomized estrogen-deprived young rats

We report the occurrence of neuronal degeneration in the supraoptic nucleus of the hypothalamus of prepubertal female rats as a consequence of ovariectomy followed by estrogen deprivation (OVX-EB). In contrast, no degenerating neurons were observed in ovariectomized rats treated with estrogens (OVX + EB) or sham-operated animals. The altered neurons in the OVX-EB group presented a cytoplasm with dilation of the rough endoplasmic reticulum lumen. The nuclear envelope also appeared dilated. There was mitochondrial swelling with disintegrated cristae, while lysosomes appeared intact. The cell nucleus showed a pattern of chromatin condensation and a nucleolus hardly distinguishable from the nucleoplasm. The neuronal alterations reported here may be due to altered gene expression in the cell nucleus resulting from induced hormonal loss during early postnatal development.

Image analysis of Feulgen-stained c-H-ras-transformed NIH/3T3 cells

Feulgen-DNA content, nuclear phenotypes, and levels of chromatin condensation were evaluated by image analysis in NIH/3T3 cells transformed with the c-H-ras oncogene of T24 cells. Three nuclear phenotypes, differing from those of untransformed control cells and defined in terms of patterns of chromatin condensation, were demonstrated microspectrophotometrically for the tumor cells. Polyploidy could only be observed in nuclei with extensive and deeply stained areas covered with condensed chromatin, i.e., only in a small fraction of the tumor cell nuclear population. The increased chromatin condensation that appeared with cell transformation affected the euchromatin zones. The image analysis provided data that, compared with those obtained in other situations involving cell transformation, could be relevant to the understanding of changes in chromatin supraorganization related to tumorigenesis and to tumor cell diagnosis.

Spermiogenesis and sperm ultrastructure in the freshwater musselDiplodon chilensis chilensis(Mollusca: Bivalvia)

Summary Spermiogenesis and sperm ultrastructure of Diplodon chilensis chilensis have been studied. Early spermatids show some peculiar characteristics which include scanty cytoplasm mitochondria restricted to a juxtanuclear region and a poorly developed Golgi complex, a feature present throughout spermiogenesis. Mid and late spermatids show an elongated nucleus and a granular pattern of chromatin condensation. The Golgi complex does not show changes associated to the formation of structures of the acrosomal complex. The mature spermatozoon agrees in all main features with those of bivalves with external fertilization. It is thus of the primitive type with bullet-shaped nucleus and five mitochondrial spheres in the middle piece. The head of the mature sperm lacks acrosomal structures. Previously reported modifications in the reproductive biology of D. ch. chilensis with respect to aspects considered representative of bivalves, have not brought about major modifications in sperm morphology.

Comparative spermatology of 11 species of Polyplacophora (Mollusca) from the suborders Lepidopleurina, Chitonina and Acanthochitonina

Transmission electron microscopy of the spermatozoa and spermatogenesis of 11 species (in three suborders Chitonina, Acanthochitonina, Lepidopleurina) of chiton has shown that each species has a sperm with a unique morphology indicating that spermatozoa can be used as a taxonomic character. Although structure is species-specific, similarities between species within suborders and subfamilies can be recognized. The spermatozoa of species from the suborders Chitonina and Acanthochitonina have a head comprising nuclear material only, the anterior portion of which is in the form of a long thin (approximately 80 nm diameter) filament. In many species the centrioles and mitochondria of the mid-piece are lateral in position, the mitochondria often being sited anteriorly alongside the nucleus. By contrast,Leptochiton asellus, a member of the more ancient suborder Lepidopleurina, has a sperm with a head comprising a nucleus and an acrosome. The mid-piece is also more conven­tional in structure with a ring of five or six spherical mitochondria (sited behind the nucleus) that surround the centrioles. The presence of the acrosome inL. asellussuggests that in the more recent chitons the acro­some has been secondarily lost. It is proposed that loss of the acrosome is correlated to a modification in egg-coat thickness. A preliminary examination of the structure of the eggs of three species has shown that those ofL. asellusare surrounded by a very thick chorion (14-30 μm) whereas inAcanthochitona crinitusandDinoplax gigasthere are regions of the chorion that are less than 2 μm thick. The morphological changes that occur during spermatogenesis are very similar in the Chitonina and Acanthochitonina. During spermiogenesis the nucleus elongates to develop a long anterior filament. Chro­matin condensation within the nucleus involves the formation of fibrils that become orientated along its long axis. Closely associated with the elongating nucleus is a manchette. InL. asellus a spherical proacrosomal vesicle appears in the spermatocytes. This vesicle becomes compressed as it matures and simultaneously it migrates to the presumptive anterior end of the spermatid where it invaginates and elongates. Although the pattern of chromatin condensation in the nucleus is similar to that described above, a manchette has not been observed.

A comparison of the structure of the spermatozoa and spermatogenesis of 16 species of patellid limpet (Mollusca: Gastropoda: Archaeogastropoda).

Light and transmission electron microscopy of the spermatozoa and spermatogenesis of 16 species (in three genera, Patella, Helcion, Cellana) of patellid limpet have shown that head lengths of the sperm range from 3 to 13 μm, and each species has a sperm with a unique morphology, indicating that the spermatozoa can be used as a taxonomic character. Although spermatozoon structure is species specific, five types can be recognized, based on the size, shape, and structure of the nucleus and acrosome. The occurrence of five morphological types of sperm, one of which (Cellana capensis) is particularly different from other patellids, suggests that the taxonomy of the family Patellidae be re-examined. The morphological changes that occur during spermatogenesis are very similar in all species, although two patterns of chromatin condensation are found. Those species with sperm that have short squat nuclei (length:breadth < 3.5:1) have a granular pattern of condensation. Species with sperm that have more elongate nuclei (length:breadth > 5:1) have an initial granular phase followed by the formation of chromatin fibrils. These fibrils become organized along the long axis of the elongating nucleus. The absence of a manchette suggests that nuclear elongation is brought about from within the nucleus.

Microheterogeneity in H1 histones and its consequences.

The extent of microheterogeneity of H1 histones in individual higher organisms, without considering post-translational modifications, is such that five to eight molecular species can be recognized. The H1 variants differ among themselves in their ability to condense DNA and chromatin fragments, and they are non-uniformly distributed in chromatin. This review assembles data that support the notion that the differences in chromatin condensation (heterochromatization) observed through the microscope are maintained by the non-uniform distribution of H1 variants, and that this pattern of chromatin condensation may determine the dynamics of chromatin during replication and may represent the commitment aspect of differentiation. The differential response of the multiple H1 variants with regard to their synthesis and turnover is consistent with this notion.

Effects of increased lifespan on chromatin condensation in the adult male housefly

Analysis of the chromatin condensation pattern in optic lobe nuclei from the adult male housefly has shown significant differences in males from a long lived, low activity (LA) group as compared to a short lived, high activity (HA) group. The rate of chromatin condensation in the LA group was very much less than that observed in the HA group, leaving the nuclei from the former group in a permanently lowered condensation state. Moreover, a detailed comparison of the amount and spatial distribution of a single high density chromatin component (HDC) suggests that the low activity conditions not only lowers the rate of chromatin condensation but also alters the normal program controlling this process. Linear discriminant analysis showed that a consistently higher percentage of nuclei from the LA group, as compared to the HA group, could be classified with a 1-day-old model derived from the HA group. These results are discussed with respect to environmental influences on lifespan and are compared to the response of lipofuscin accumulation in low activity male houseflies.

Nuclear morphogenesis during spermiogenesis in the nudibranch molluscHypselodoris tricolor (Gastropoda, opisthobranchia)

AbstractAn electron microscope study was carried out on Hypselodoris tricolor spermatids to describe the development of the nuclear morphogenesis and investigate the possible cause(s) of the change in the shape of the spermatid nucleus during spermiogenesis. Three different stages may be distinguished in the course of the nuclear morphogenesis on the basis of the morphology and inner organization of the nucleus. Stage 1 spermatid nuclei are spherical or ovoid in shape and the nucleoplasm finely granular in appearance. Stage 2 nuclei exhibit a disc‐ or cup‐shaped morphology, and the chromatin forms short, thin filaments. During stage 3, a progressive nuclear elongation takes place, accompanied by chromatin rearrangement, first into fibers and then into lamellae, both formations helically oriented. A row of microtubules attached to the nuclear envelope completely surrounds the nucleus. Interestingly, the microtubules always lie parallel to the chromatin fibers adjacent to them. Late stage 3 spermatids show the highest degree of chromatin condensation and lack the manchette at the end of spermiogenesis. Our findings indicate the existence of a clear influence exerted on the chromatin by the manchette microtubules, which appear to be involved in determining the specific pattern of chromatin condensation in Hypselodoris tricolor.

Age-related chromatin condensation in flight muscle nuclei of the adult male housefly, Musca domestica

Computer analysis of indirect flight muscle nuclei from the adult male housefly, M. domestica, has shown significant change with age in the chromatin condensation pattern. The pattern was analyzed by examining low, medium, and high density chromatin components (LDC, MDC, HDC). No significant change occurred in HDC with age, and the amount and distribution of LDC and MDC remain relatively stable between Day 1 and Day 4 post-eclosion. However, the analysis showed a significant increase in the amount of MDC with a corresponding decrease in the amount of LDC between Day 4 and Day 14. This exchange was accompanied by a significant redistribution of both components. These results are discussed with reference to the biochemical and ultrastructural profile of the flight muscle with age, and to age-related changes in the condensation pattern of specific brain and Malpighian tubule nuclei described earlier.

Shaping of the sperm nucleus in Marsilea: A distinction between factors responsible for shape generation and shape determination

Spermiogenesis in Marsilea vestita involves the elongation of a roughly spherical nucleus into a spiral that is composed of four to five gyres. A ribbon of microtubules is associated with the outer edge of the nucleus throughout the shaping process. In order to observe nuclear morphogenesis in the absence of microtubules, developing microspores were treated with drugs that are known to affect microtubule assembly. Spermatids cultured in the presence of colchicine from the beginning of spermiogenesis do not form a microtubule ribbon. The nuclei of these cells change from a spherical to an irregular shape with elongate branches or loops. The normal spiral nucleus and elongate rod of condensed chromatin are not formed and the pattern of chromatin condensation is also abnormal. These observations indicate that in Marsilea microtubules do not provide the mechanical force for nuclear shape generation. Bulk chromatin condensation can also be eliminated as the force behind nuclear shaping, because during normal development the chromatin condenses only after nuclear shaping is well advanced. We suggest that a force-generating system is located near or is a part of the nuclear envelope. Microtubules may, however, be important in the determination of the final shape of the nucleus either by organizing or directing the force-generating system or by externally restricting or guiding the shaping nucleus. Microtubules may also function in controlling the pattern of chromatin condensation.

Aspects of the structure and development of monotreme spermatozoa and their relevance to the evolution of mammalian sperm morphology.

The elongated spermatid nuclei of monotremes exhibit a circumferentially arranged spiral pattern of chromatin condensation, and ultimately form helical filiform sperm heads up to 50 microns long and either circular or slightly oval in transverse section. The acrosome is formed by the collapse of the proacrosomal vacuole onto the rostral surface of the elongating nucleus. However, genesis of acrosomal material occurs in the absence of a prominent proacrosomal granule. The flagellum becomes inserted into the distal extremity of the nucleus, the most proximal mitochondria of the midpiece directly abutting the nuclear membrane, so that a prominent neck region is absent. The axoneme is simple and, in the midpiece, small dense peripheral fibres are closely applied to the outer surface of each of the nine microtubule doublets. The cortical fibrous sheath of the principal piece is an anastomosing spiral that lacks lateral columnar elements. The spermatozoal cytoplasmic droplet undergoes migration and is lost during epididymal passage. Monotreme spermatozoa exhibit a montage of features, some of these being also found in marsupials and some in sauropsidan vertebrates, as well as a number of their own distinctive features. It is concluded that monotreme spermatozoa also have a close affinity with the unspecialised spermatozoa of some eutherian mammals.

Fluorimetric measurements and chromatin condensation patterns of nuclei from 3T3 cells throughout G1.

Using two cytological methods based on nuclear morphology, quinacrine dihydrochloride (QDH) staining and premature chromosome condensation (PCC), it has been possible to identify cell cycle positions within G1 of growing and arrested 3T3 cells. The fluorescent intensity of QDH-stained interphase cells appears to decrease as the cells pass from mitosis to S phase. Likewise, the length and thickness of prematurely condensed chromatids can be related to the cells; position within the G1 period. Data are presented that deal with three interrelated topics: 1) We determined by fluorometric measurements of nuclei from 3T3 cells that the visual observation of the decrease in QDH fluorescence during G1 reflects an actual decrease in total fluorescence and not a dispersion of the fluorescent chromatin in a larger nuclear area. 2) We correlated the results obtained by QDH staining with those of PCC on the same cell samples blocked in G1 by different conditions. Serum-starved and contact-inhibited cell nuclei had the highest intensity, hydroxyurea-treated ones had the lowest intensity, while that of isoleucine-deprived cells was in between. The same relative order of G1 positions was obtained based on PCC morphology. Thus, both methods monitor the state of chromatin condensation and can be used to identify cell cycle position within G1. 3) We showed with both methods that the states of chromatin resulting from the various G1 blocking conditions differ from each other.

Ultrastructural differentiation of rat Sertoli cells.

Fine structural cytodifferentiation of the Sertoli cells of the rat testis was examined at 2 week intervals from Day 7 postnatally to sexual maturity. The cells undergo rapid morphological differentiation during the first 5 weeks after birth. These changes include a large increase in cell size and in the extent of cytoplasmic processes between germinal cells; changes occur in the pattern of nuclear chromatin condensation and nuclear envelope infoldings and in the development of cellular organdIes, particularly the smooth endoplasmic reticulum, Golgi apparatus and mitochondria. Differentiation of the basal cytoplasm consists primarily of a regional accumulation of microtubules and inicrofilaments, whereas apical surface specializations consist of fingerlike cytoplasmic projections. An apparent sequence of events was observed in the development of the junctional complexes between neighboring cells and in the formation of the tripartite nucleolus. This Sertoli cell structural differentiation occurring during the prepuberal period correlates with the appearance of various physiologic and metabolic changes in the function of the developing testis.

The cellular anatomy of embryos of the nematode Caenorhabditis elegans: Analysis and reconstruction of serial section electron micrographs

As described from light microscopy, embryogenesis of the free-living soil nematode Caenorhabditis elegans follows a strictly determinate cleavage pattern, producing a newly hatched juvenile with about 550 cells arranged quite predictably. In this communication we present results on the electron microscopy of C. elegans embryos and introduce methods for fixing, embedding, and serially sectioning embryos encased in the egg shell. Fixation at elevated temperature either with osmium tetroxide alone or with glutaraldehyde followed by osmium tetroxide gives reproducible results with embryos in all developmental stages so far tested, from the fertilized egg to hatching. Eighteen wild-type eggs at various stages have been sectioned to date. We have achieved using newly developed procedures for analyzing electron micrographs of serial sections detailed reconstructions of the cellular anatomy of complete embryos of a metazoan organism. Three-dimensional serial section reconstructions were made with a computer system. We characterize and map the 24 cells of an early-stage embryo in this report. Additionally, we can specify the lineage history of all cells of this embyro by matching the reconstructed three-dimensional arrangement of this series to a living embryo at this stage, where cell lineage has been observed with Nomarski optics and analyzed using videotape ( U. Deppe, E. Schierenberg, T. Cole, C. Krieg, D. Schmitt, B. Yoder, and G. von Ehrenstein, 1978, Proc. Nat. Acad. Sci. USA 75, 376–380). In addition, cytoplasmic and nuclear morphological features such as incomplete membranes between sister cells, rounding-off of the cytoplasm, and chromatin condensation patterns have been correlated with cell division. Mapping of such structures presents a new method by which supplementary lineage information can be obtained directly from an electron micrographic series.

The ultrastructure of immature Sertoli cells. Maturation-like changes during culture and the maintenance of mitotic potentiality.

Cell aggregates dissociated from 10-day-old rat testes form epithelial colonies in culture. These epithelial cells have been identified as supporting cells (immature Sertoli cells) by the close similarity between the nuclear and cytoplasmic structures of the cultured cells and those of supporting cells in vivo. The cultured supporting cells undergo maturation-like changes in serum-enriched media containing follicle stimulating hormone (FSH). These changes include a change in the pattern of chromatin condensation, the development of large nucleoli and nuclear infoldings, a progressive predominance of the smooth over the rough endoplasmic reticulum and the appearance of luminal (apical) differentiations. The differentiations of the luminal surface consist of finger-like cytoplasmic extensions, a regional accumulation of microfilaments and microtubules and the junctional complexes between neighboring cells near the lumen. In the presence of FSH there is a low but sustained mitotic activity in the population of cultured supporting cells. This FSH-dependent mitotic activity was observed from Day 4-15 in culture. It is suggested that while several structural differentiations of the supporting cells develop under these conditions in Vitro, the luminal region of these cells does not differentiate in the same way as the apical region of Sertoli cells in vivo.

Nuclear and Chromosomal Changes During Sperm Formation in the Scorpion,Centruroides Vittatus(Say)

SUMMARYThe pattern of chromatin condensation in the developing sperm nucleus of the scorpion, Centruroides vittatus, is described. The chromatin fibrils become especially distinct and remain well defined until quite late in spermiogenesis. The fibrils that compose the chromosomes are arranged in groups. The chromosomes in this species have diffuse kinetochores. The sperm head is helical in the swimming sperm and acquires this shape without the influence of microtubules, which are not present. Also absent is the centriole adjunct, though this organelle is found in other scorpions.

Influence of infra-optimal breeding temperature on spermiogenesis of the locust Locusta migratoria: II. Abnormalities in differentiation of the nucleus

The sterile males of the Migratory locust that have been raised under infra-optimal temperature conditions produce spermatids with highly abnormal nuclei. These are spherical in young spermatids but later they do not elongate uniformly and they show deep invaginations into which cytoplasmic organelles penetrate. Condensation of the chromatin also is disturbed and the typical honeycomb pattern of the locust spermatid is never achieved. The most advanced stage of condensation that can be reached corresponds to stage 6 of the normal process. Nuclei demonstrating a “stage 6” pattern of chromatin condensation are never thin and slender, as they normally should be at this stage. The shape of the nucleus in these experimental animals seems to be correlated with the pattern of perinuclear microtubules rather than with the state of chromatin condensation. Nuclear abnormalities as well as those regarding the cytoplasmic organelles are compared to several spermiogenetic defects reported in various sterile mutants of Drosophila or in insects submitted to heat-shocks. A total sterility at temperatures only slightly inferior to the usual breeding ones has never yet been reported in insects other than locusts; temperature is thus demonstrated to be a very critical factor for fertility not only when it is too high but also when it does not reach an optimal level.

Structural changes in chromatin during interphase

The structural organization of the dense chromatin in G1, S and G2 of onion meristem cells has been evaluated. A naturally synchronous subpopulation of caffeineinduced binucleate cells was employed. — Fibre size is positively correlated with cell stage in interphase. Fibre size distribution is unimodal in G1 nuclei with the peak at 12.5 nm in diameter, while in G2 the distribution is bimodal due to a new population of thicker fibres (22.5 nm in diameter). Separation between fibre centres takes place between mid G1 and mid S. — Using stereological principles, the length of chromatin fibres integrated in the chromatin patches could be estimated. This length remains constant from mid G1 to mid S, when a 1.5 fold increase in total DNA takes place. On the other hand, 40 mm/hour of chromatin fibre is integrated in the chromatin patches between mid S and mid G2. Comparable data of the relative proportion of the different nuclear components have been obtained in each interphase period. — The reported changes provide evidence of a cyclic pattern of chromatin condensation, which may be the structural support for a model of chromatin function in these cycling cells.