Degree Of Chromatin Condensation Research Articles

Epigenetics and Noncoding RNA – Principles and Clinical Impact

AbstractGenetics studies the inheritance of genetic information encoded by the base pair sequence and its variants. Sequence variants can have severe consequences as seen in genetically inherited diseases (e. g. osteogenesis Imperfecta, hypophosphatasia). On the other hand, epigenetics deals with inherited and dynamically reversible modifications of chromatin without changing the base pair sequence, resulting in a change in phenotype without a change in genotype. These modifications primarily exert their effects by influencing gene expression. Initially, the definition of epigenetics exclusively comprised inherited changes that persist across several generations without changes in the DNA sequence. This definition has been extended to include also dynamic and partially reversible changes that occur more short-term. These gene modulatory effects introduce new levels of complexity and are crucial determinants of cell fate and organismal development. With its length of approximately two meters, human DNA has to be compacted to fit into the nuclei and fulfill its functions. DNA is wrapped around histone octamers into so-called nucleosomes. DNA, histones, and other DNA-associated proteins together form what is called chromatin. DNA packaging is achieved by variable degrees of chromatin condensation depending on cell type and context. Epigenetic transcriptional regulation modifies the affinity and accessibility of cis-regulatory elements (CREs) for transcription factors and the basic transcriptional machinery and governs interaction between CREs. CREs include promoters, enhancers, silencers, and insulators and are potent modulators of gene expression impacting core cell biological processes such as proliferation and differentiation. Chromatin looping and remodeling by differential covalent modifications of DNA (e. g., methylation or hydroxylation) and histone tails (e. g., acetylation or methylation) elicit fundamental changes in CRE accessibility, thus impacting gene expression. Chromatin looping depends on a specialized machinery including cohesins. Chromatin modifications are mediated by specific enzymes like DNA methylases (DNMTs), histone-modifying enzymes, like histone methyl- and acetyltransferases (KMTs, HATs/KATs), and histone demethylases and deacetylases (KDMs, HDACs). It becomes increasingly evident that epigenetic (dys)regulation plays a decisive role in physiology and pathophysiology, impacting many age-related diseases like cancer and degenerative pathologies (e. g., osteoporosis, Alzheimer’s, or Parkinson’s) in a significant fashion. Recently, small-molecule inhibitors of chromatin-modifying enzymes (e. g., vorinostat) have been identified and successfully introduced in therapy. Significant progress in high-throughput sequencing technologies and big data analysis has broadened our understanding of noncoding (nc) RNAs and DNA sequence regions in (post-)transcriptional regulation and disease development. Among ncRNAs that play vital roles in gene expression are micro- (miRs) and long noncoding RNAs (lncRNAs; e. g., XIST or HOTAIR). By interacting with the coding genome, these RNAs modulate important genetic programs. Interfering RNAs can, for example, enhance the post-transcriptional degradation of transcripts, altering their translation, or assist in the recruitment of chromatin-modifying enzymes to regulate transcription. They can also be packaged into extracellular vesicles as cargo and thus deliver critical information to the microenvironment or even systemically to distant tissues. Therefore, ncRNAs represent a novel playground for therapeutical investigations and supplement epigenetic mechanisms of gene regulation while being subject to epigenetic regulation themselves. Last but not least, dysregulated ncRNAs can also propagate disease. Until recently, the detection of epigenetic phenomena necessitated invasive diagnostic interventions. However, with the arrival of so-called “liquid biopsies” an analysis of circulating cell-free DNA fragments (cfDNA) and RNAs as well as vesicle-packed RNAs through minimal invasively drawn blood samples can be obtained. Such “fragmentomics” and RNAomics approaches on peripheral blood will ultimately serve as diagnostic tools for personalized clinical interventions.

Hormone Concentrations and Semen Parameters in Criollo Breed Stallions Under Training

The stress associated with training may reduce reproductive efficiency in Criollo stallions. The objective of this study was to compare semen quality and hormone concentrations in Criollo stallions under training or under regular field conditions. Criollo breed stallions (n = 18) were evaluated during the spring. The exercise group (n = 9) performed 1 hour of exercise per day and participated in competitions during the experimental period. The control group (n = 9) neither performed exercise nor participated in competitions. Serum and semen samples were obtained every 15 days (two separate ejaculates an hour apart). Sperm motility, velocity, and morphology were evaluated with a phase-contrast microscope and concentration by a hemocytometer. Diff-Quik stain was used to identify polymorphonuclear cells, and the degree of chromatin condensation was evaluated with the toluidine blue stain. The sperm survival test was performed at a room temperature of 22°C. Semen evaluation was performed in raw samples and in samples diluted in a skim milk and glucose-based extender. Cortisol, testosterone, and estradiol were measured using radioimmunoassay. There was no effect of exercise on testosterone and estradiol concentrations (P = .28 and P = .97, respectively). However, in the exercise group, cortisol concentration was higher after exercise (P = .004). There was an effect of exercise on the following semen parameters: gel-free volume (P < .001), sperm motility (P < .0001), total number of sperm (P = .0001), normal sperm morphology (P < .0001), and total number of morphologically normal and motile sperm (P < .001). No effect of exercise was found in the following semen parameters: color, pH, and sperm concentration. This study showed that exercise had a negative impact on seminal quality; nevertheless, semen parameters were within the normal ranges established for the equine species.

Air-Drying Llama Sperm Affects DNA Integrity.

The objective of this study was to evaluate the effects of air-drying preservation on llama sperm DNA. Semen collections were carried out using electroejaculation under general anesthesia. A total of 16 ejaculates were processed from 4 males (n = 4, r = 4). Each sample was diluted 4:1 in a collagenase solution in TALP media, then incubated and centrifuged at 800 g for 8 min. The pellet was re-suspended to a concentration of 20 million sperm/ml in TALP. Then the samples were placed onto sterile slides forming lines and were left to dry under laminar flow for 15 min. After this, the slides were placed into Falcon centrifuge tubes and kept at 5°C. Sperm characteristics (motility, membrane function, viability and morphology) were evaluated in raw semen and in the air-dried samples kept at 5°C for 30 min. DNA evaluation (integrity and degree of chromatin condensation) was carried out in raw semen and in the air-dried samples after 30 min, 7, 14, 21, 30, and 60 days after preservation. To compare raw semen to the air-dried samples, a Wilcoxon test was used for all sperm characteristics except for DNA, where a paired Student t-test was applied. A split plot design was used to compare chromatin condensation between the different periods of preservation and a Kruskal Wallis test was used to compare DNA integrity. Motility, membrane function, viability and sperm with intact DNA decreased in the air-dried samples (p < 0.05), while morphology and chromatin condensation were not affected (p > 0.05). No significant differences were observed in the percentage of sperm with condensed chromatin between the different periods of preservation (p > 0.05). On the other hand, a significant decrease in the percentage of sperm with intact DNA was observed as from day 7 of preservation (p < 0.05). In conclusion the air-drying process has a negative effect on llama sperm DNA, hence the media used will need to be improved to protect DNA and be able to implement this technique in this species.

Thermodynamic Study of Interactions of Distamycin A with Chromatin in Rat Liver Nuclei in the Presence of Polyamines.

We studied the thermodynamics of melting of isolated rat liver nuclei with different degrees of chromatin condensation determined by the concentration of polyamines (PA) and the solution ionic strength, as well as the effect of the antibiotic distamycin A (DM) on melting. Differential scanning calorimetry (DSC) profiles of nuclear preparations contained three peaks that reflected melting of three main chromatin domains. The number of peaks did not depend on the degree of condensation; however, nuclei with more condensed chromatin had a higher total enthalpy. DM stabilized peaks II and III corresponding to the melting of relaxed and topologically strained DNA, respectively, but destabilized peak I corresponding to the melting of nucleosome core histones. At the saturating concentration (DM/DNA molar ratio = 0.1), DM increased Tm of peaks II and III by ~5°C and decreased Tm of peak I by ~2.5°C. Based on the dependence of ΔH on DM concentration, we established that at low DM/DNA ratio (≤0.03), when DM interacted predominantly with AT-rich DNA regions, the enthalpy of peak II decreased in parallel with the increase in the enthalpy of peak III, which indicated that DM induces structural transitions in the nuclear chromatin associated with the increase in torsional stress in DNA. An increase in free energy under saturation conditions was equal to the change in the free energy of DM interaction with DNA. However, the increase in the enthalpy of melting of the nuclei in the presence of DM was much greater than the enthalpy of titration of nuclei with DM. This indicates a significant increase in the strength of interaction between the two DNA strands apparently due, among other things, to changes in the torsional stress of DNA in the nuclei. Titration of the nuclei with increasing PA concentrations resulted in the decrease in the number of DM-binding sites and the non-monotonous dependence of the enthalpy and entropy contribution to the binding free energy on the PA content. We suggested that the observed differences in the thermodynamic parameters were due to the different width of the minor groove in the nuclear chromatin DNA, which depends on PA concentration.

Condensation patterns of prophase/prometaphase chromosome are correlated with H4K5 histone acetylation and genomic DNA contents in plants.

Mitotic prophase chromosome condensation plays an essential role in nuclear division being therefore regulated by highly conserved mechanisms. However, degrees of chromatin condensation in prophase-prometaphase cells may vary along the chromosomes resulting in specific condensation patterns. We examined different condensation patterns (CPs) of prophase and prometaphase chromosomes and investigated their relationship with genome size and distribution of histone H4 acetylated at lysine 5 (H4K5ac) in 17 plant species. Our results showed that most species with small genomes (2C < 5 pg) (Arachis pusilla, Bixa orellana, Costus spiralis, Eleutherine bulbosa, Indigofera campestris, Phaseolus lunatus, P. vulgaris, Poncirus trifoliata, and Solanum lycopersicum) displayed prophase chromosomes with late condensing terminal regions that were highly enriched in H4K5ac, and early condensing regions with apparently non-acetylated proximal chromatin. The species with large genomes (Allium cepa, Callisia repens, Araucaria angustifolia and Nothoscordum pulchellum) displayed uniformly condensed and acetylated prophase/prometaphase chromosomes. Three species with small genomes (Eleocharis geniculata, Rhynchospora pubera, and R. tenuis) displayed CP and H4K5ac labeling patterns similar to species with large genomes, whereas a forth species (Emilia sonchifolia) exhibited a gradual chromosome labeling, being more acetylated in the terminal regions and less acetylated in the proximal ones. The nucleolus organizer chromatin was the only chromosomal region that in prometaphase or metaphase could be hyperacetylated, hypoacetylated or non-acetylated, depending on the species. Our data indicate that the CP of a plant chromosome complement is influenced but not exclusively determined by nuclear and chromosomal DNA contents, whereas the CP of individual chromosomes is clearly correlated with H4K5ac distribution.

Ultrastructural organization of the domains in the cell nucleus of dicotyledonous and monocotyledonous plants under abiotic stress

The transformation of the structural organization of interphase nuclei of a plant cell, depending on the type of tissue, ploidy, and the action of abiotic factors of the medium, has been studied by light and transmission electron microscopy. It is shown that the location and the quantitative relationship between condensed and decondensed chromatin, the presence and localization of nuclear bodies in the plant nucleus, and the presence of invaginations of the nuclear membrane and inclusions depend on the tissue type and cell age as well as the intensity, time, and type of exposure to abiotic factors. Examples of different degrees of chromatin condensation in one plant organism are given. It has been established that the degree of chromatin condensation and decondensation, the state of other domains and nonspecific inclusions of the cell nucleus can be artificially modeled for research purposes or subsequent modification. The issue of application of such technologies for the creation and selection of stable forms of agricultural plants taking into account the controlled modification of their genome is discussed. A possible mechanism for incorporation of nonspecific inclusions in a nuclear compartment is proposed.

Abstract A45: Chromatin dynamics of the zebrafish retina during development and aging

Abstract During organogenesis, the retina develops from a single sheet of undifferentiated neuroepithelium into a layered mature tissue composed of Müller, photoreceptor, horizontal, bipolar, amacrine, interplexiform and ganglion cells. This differentiation of various retinal cell types requires the transcription of distinct sets of genes in precise temporal orders. It is unclear how chromatin organization and modifications are specifically regulated at global and regional levels to facilitate the generation of distinct transcriptional profiles required for different retinal cell types. Nor do we know to what extent chromatin structure and organization are altered during retinal aging, or how such alterations lead to unbalanced gene expression profiling and subsequent cell degeneration. In this study, we analyzed the dynamic characteristics of nuclear structures and chromatin organizations of zebrafish retinal cells during development and aging. We show that the degrees of chromatin condensation vary significantly among different retinal cell types, as suggested by varying nuclear volumes and heterochromatin/euchromatin organization. These structural features are altered during the aging process as well. For example, the nuclear volumes of cone photoreceptors increase significantly during aging. The distribution patterns of H3K9me3 and H4K20me3 heterochromatin modifications also vary dramatically among different retinal cell types and at different ages. The gross alterations in nuclear structures and chromatin condensation may suggest extensive changes in gene expression activities. These findings provide insight into how chromatin organization and modifications underlie balanced global transcriptional profiles and consequently, the overall fitness of retinal cells, as well as how such regulations might be altered during the aging process. Our study may have significant social impact because with increasing life expectancy, more and more older people will suffer from age-related macular degeneration and the identification of proper interventions to slow down the retinal aging process would dramatically enhance quality of the life for the elderly. Citation Format: Xiangyun Wei. Chromatin dynamics of the zebrafish retina during development and aging. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A45.

Small human sperm vacuoles observed under high magnification are pocket-like nuclear concavities linked to chromatin condensation failure

Since an embryo’s ability to grow to the blastocyst stage and implant can be improved by selection of a normal spermatozoon with a vacuole-free head, this study set out to determine the nature of small sperm vacuoles observed under high magnification (>×6300). For 15 infertile men with various sperm profiles, high-magnification microscopy was used to select motile, morphometrically normal spermatozoa with no vacuoles (n=450) or more than two small vacuoles (each of which occupied less than 4% of the head’s area; n=450). Spermatozoa acrosome reaction status and degree of chromatin condensation were analysed. Three-dimensional deconvolution microscopy was used to accurately image the nucleus and acrosome at all depths in all spermatozoa. In all 450 spermatozoa with small vacuoles, the latter were seen to be abnormal, DNA-free nuclear concavities. Spermatozoa with small vacuoles were significantly more likely than vacuole-free spermatozoa to have noncondensed chromatin (39.8% versus 9.3%, respectively; P<0.0001). There was no significant difference between the two groups of spermatozoa in terms of acrosome reaction status. No association between chromatin condensation and acrosome reaction status was observed. Small human sperm vacuoles observed under high magnification are pocket-like nuclear concavities related to failure of chromatin condensation.The present study set out to determine the nature of small sperm vacuoles observed under high magnification (>×6300), since these structures reportedly have a harmful effect on embryo development and implantation. For 15 infertile men with various sperm profiles, high-magnification microscopy was used to select a total of 900 motile, morphometrically normal spermatozoa with no vacuoles (n=450) or more than two small vacuoles (each of which occupied less than 4% of the head’s area; n=450). We subsequently analysed the spermatozoa’s acrosome reaction status and degree of chromatin condensation. Three-dimensional deconvolution microscopy was used to accurately image both the nucleus and the acrosome at all depths in each of the 900 spermatozoa. In all 450 spermatozoa with small vacuoles, the latter were seen to be abnormal, DNA-free nuclear concavities. Spermatozoa with small vacuoles were significantly more likely than vacuole-free spermatozoa to have non-condensed chromatin (39.8±2.4% versus 9.3±1.3%, respectively; P<0.0001). There was no significant difference between the two groups of spermatozoa in terms of the acrosome reaction status. No association between chromatin condensation and acrosome reaction status was observed. Small human sperm vacuoles observed under high magnification are pocket-like nuclear concavities related to the failure of chromatin condensation.

Amplification of human DNA for probing DEB‐induced damage via a quantitative polymerase chain reaction

Diepoxybutane (DEB) is a bifunctional alkylating agent linked to the high incidence of certain cancers in industrial workers exposed to butadiene. DEB forms interstrand cross‐links in duplex DNA, inhibiting replication and transcription. Since PCR mimics DNA replication, we can use it to assess cross‐linking by measuring the amount of DNA available for amplification. Our ultimate goal is to determine whether DNA structure affects DEB reactivity in vivo by monitoring damage through a quantitative polymerase chain reaction (PCR) at regions that differ in their degree of chromatin condensation. To date, we have optimized the conditions for traditional PCR within human DNA at nuclear loci of about 10 kb in length. We have also obtained CT values, using real‐time PCR with SYBR green dye. When our real‐time PCR conditions are optimized, we can use the difference between amplification of control, unmodified DNA versus DNA from DEB‐treated cells to determine the lesion frequency at the loci of interest. These findings are important because they will further characterize the mode of action of these compounds and thus help determine the carcinogenic risk for workers in the butadiene industry. This project was supported by grants from the National Center for Research Resources (5P20RR016463–12) and the National Institute of General Medical Sciences (8 P20 GM103423–12) from the NIH.

Dermatan sulfate synergizes with heparin in murine sperm chromatin decondensation

The mammalian sperm nucleus contains an unusually condensed chromatin, due to replacement of the majority of histones by protamines. However, soon after the spermatozoon penetrates the ooplasm at fertilization, decondensation of this densely packed chromatin must occur to allow formation of the male pronucleus and syngamy. Decondensation is accomplished by protamine disulfide bond reduction by oocyte glutathione and replacement of protamines by oocyte histones with the aid of an acceptor molecule. Previous results from our laboratory have demonstrated that heparan sulfate (HS) present in the ooplasm functions as protamine acceptor during human sperm decondensation in vivo. In the present paper, we analyze the role of heparin, structural analogue of HS, and dermatan sulfate (DS) in murine sperm chromatin decondensation in vitro, including the possibility of a synergistic effect between both glycosaminoglycans. Decondensation was assessed under phase contrast microscopy following incubation of murine spermatozoa with glutathione and either heparin, DS, or a combination of both. Ultrastructural changes taking place during decondensation were analyzed by transmission electron microscopy. Both glycosaminoglycans were able to promote the decondensation of murine spermatozoa in vitro but the decondensing ability of heparin was significantly higher. Use of both glycosaminoglycans together revealed the existence of a synergistic effect. Transmission electron microscopy analysis of decondensing spermatozoa supported these findings. Synergism between heparin and DS was observed both in capacitated and non-capacitated spermatozoa but decondensation kinetics was faster in the former. The results obtained indicate a new potential role for dermatan sulfate in murine sperm decondensation at fertilization and provide evidence of differences in the degree of chromatin condensation throughout the murine sperm nucleus.

Arrangement of nuclear structures is not transmitted through mitosis but is identical in sister cells

Although it is well known that chromosomes are non-randomly organized during interphase, it is not completely clear whether higher-order chromatin structure is transmitted from mother to daughter cells. Therefore, we addressed the question of how chromatin is rearranged during interphase and whether heterochromatin pattern is transmitted after mitosis. We additionally tested the similarity of chromatin arrangement in sister interphase nuclei. We noticed a very active cell rotation during interphase, especially when histone hyperacetylation was induced or transcription was inhibited. This natural phenomenon can influence the analysis of nuclear arrangement. Using photoconversion of Dendra2-tagged core histone H4 we showed that the distribution of chromatin in daughter interphase nuclei differed from that in mother cells. Similarly, the nuclear distribution of heterochromatin protein 1β (HP1β) was not completely identical in mother and daughter cells. However, identity between mother and daughter cells was in many cases evidenced by nucleolar composition. Moreover, morphology of nucleoli, HP1β protein, Cajal bodies, chromosome territories, and gene transcripts were identical in sister cell nuclei. We conclude that the arrangement of interphase chromatin is not transmitted through mitosis, but the nuclear pattern is identical in naturally synchronized sister cells. It is also necessary to take into account the possibility that cell rotation and the degree of chromatin condensation during functionally specific cell cycle phases might influence our view of nuclear architecture.

Ultrastructural study of spermatogenic stages in the protandrous sparid fish Diplodus cervinus cervinus (Lowe, 1838) from the South Eastern Mediterranean coast

The current study was designed to study the ultrastructure of the spermatogenic stages of the protandrous hermaphrodite sparid Diplodus cervinus cervinus. Although, it is a useful tool to enhance understanding of germ cells differentiation in this economic species, none of the available references paid attention to the studied species. The testis of the studied specie is tubular in shape and the germ cells are arranged in cysts or clusters within the seminiferous lobules. Spermatogenesis occurs in several places along the length of each lobule and induced by the action of the somatic steroidogenic secretory cells which are known as Leydig cells. Such cells contained four main morphological structural characteristics a vesicular nucleus, ovoid and elongated mitochondria with tubular cristae, a number of smooth endoplasmic reticula, and a considerable amount lipid droplets in the cytoplasm. Spermatogenic cyst displays round shaped cells with large nuclei containing clumps of heterogenic dense chromatin and reduced cytoplasm known as primary spermatogonia. They undergo a series of mitotic divisions to reach the secondary spermatogonia stage; such cells irreversibly divide meiotically to form primary and secondary spermatocytes. Spermatids are seen in different stages of spermiogenesis, as indicated by the degree of chromatin condensation. They underwent a shape remodeling and a size reduction during spermiogenesis as the morphology of the spermatid nucleus gradually changed and several mitochondria and centrosomes moved to a position just behind the nucleus of the spermatid. Spermatozoan ultrastructure showed that it is anacrosomal primitive teleostean type (type I) with round head, the posterior part of the nucleus is indented a nuclear fossa which displays a bell-shaped in longitudinal section and circular in transverse section containing centeriolar complex, part of the basal body and cytoplasm. The chromatin is heterogeneously granular, high electron-dense containing tightly packed fibers. The mid piece is short containing mitochondrial ring which consists of spherical or ovoid mitochondria. The axoneme consists of nine double outer tubules and single microtubular constructions. In conclusion, the testis structure could be described as the unrestricted spermatogonial testicular type. More attention should be rewarded to the female of this species in order to give a clear view about the process of oogenesis. Key words: Spermatogenesis, Sparid, protandrous, hermaphrodite, anacrosomal, primitive teleostean spermatozoa.

Phosphorylation of Histone H3S10 in Animal Chromosomes: Is There a Uniform Pattern?

Phosphorylation of serine 10 in histone H3 (H3S10ph) has been extensively analyzed and appears to be a conserved chromatin change associated with chromosome condensation in different eukaryotic organisms. In this work, we report the distribution of H3S10ph during meiosis in monocentric and holokinetic chromosomes of 6 insect species and in mitotic chromosomes of 7 mammalian species, aiming to investigate the labeling patterns in phylogenetically distant groups. The results indicated a very similar phosphorylation timing and distribution pattern among insects. The sex chromosomes of insects analyzed were always undercondensed and hypophosphorylated. Similarly, the micro chromosomes of the bug Pachylis aff pharaonis were also undercondensed and hypophosphorylated. Holokinetic chromosomes of bugs and monocentric chromosomes of grasshoppers and beetles displayed identical phosphorylation pattern in spite of the difference in the centromere type. Among mammals, a uniform chromosome phosphorylation was observed in marsupials, whereas bat chromosomes displayed a longitudinal banding pattern. These data indicate that, in general, the intensity of H3S10 phosphorylation in animal chromosomes is variable among the distinct chromosome types and associated with the degree of chromatin condensation at metaphase, but it may vary between different groups of animals.

Large human sperm vacuoles observed in motile spermatozoa under high magnification: nuclear thumbprints linked to failure of chromatin condensation

An embryo's ability to grow and implant can be improved by selection of a normal spermatozoon with a vacuole-free head. However, large vacuoles in spermatozoa have yet to be fully characterized. The present study aimed to determine whether these vacuoles are of nuclear, membrane and/or acrosomal origin. We studied 15 infertile patients with differing sperm profiles. For each sperm sample, we used high-magnification (×10 000) contrast microscopy to select and assess 30 normal 'top' spermatozoa and 30 spermatozoa with a large sperm-head vacuole (≥ 25% of the head's cross-sectional area). We subsequently analysed the spermatozoa's degree of chromatin condensation (aniline blue staining), DNA fragmentation (terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling assay) and chromosome content (fluorescence in situ hybridization X,Y,18). Atomic force microscopy enabled us to map the plasma sperm membrane in detail. Three-dimensional deconvolution microscopy enabled us to reconstruct images of the nucleus and acrosome in 'top' and 'vacuolated' spermatozoa. We studied a total of 450 'top' spermatozoa and 450 vacuolated spermatozoa. The rate of non-condensed chromatin was higher for 'vacuolated' spermatozoa than for 'top' spermatozoa (36.2 ± 1.9 versus 7.6 ± 1.3%, respectively; P < 0.0001). 'Top' and 'vacuolated' spermatozoa did not differ significantly in terms of DNA fragmentation (0.7 ± 0.4 versus 1.3 ± 0.4% respectively; P = 0.25) or aneuploidy (1.1 ± 0.5 versus 2.2 ± 0.7% respectively; P = 0.21). The majority of aneuploid spermatozoa (9 out of 15) lacked chromatin condensation. In all vacuolated spermatozoa, the acrosome was intact, the plasma membrane was sunken but intact and the large vacuole was identified as an abnormal, 'thumbprint'-like nuclear concavity covered by acrosomal and plasmic membranes. The large vacuole appears to be a nuclear 'thumbprint' linked to failure of chromatin condensation.

Modifications in the oviducts of workers and queens of Melipona quadrifasciata anthidioides (Hymenoptera: Apidae) with different ages

The study of morphological features of highly eusocial bees has helped to answer a series of questions concerning their biology. The labor division places the females into two castes, queen with reproductive function and worker with a wide variety of tasks. However, in different species and under different conditions, workers can develop ovaries and lay trophic eggs that are eaten by the queen or used to originate males. In this work, the development of the oviducts was monitored in workers and virgin queens of Melipona quadrifasciata anthidioides specimens of different ages to verify whether there is permanent sterility of these queens and workers due to aging. Lateral and common oviducts of virgin queens aged <7, 10, 15, 20, and 25days old, physogastric queens, nurse, and forager workers were analyzed for histology and ultrastructure. Although the structural organization of the lateral and common oviducts were very similar, differences in width of the cuticle and the degree of chromatin condensation were observed, indicating differences in the development of this organ of the reproductive system between the castes. It was also demonstrated that electron-lucent vesicles appear to be related to the formation of the cuticle that lines the oviducts. Because no cellular death characteristics were found, it can be inferred that the absence of mating of the queens as old as of 25days of age does not cause permanent sterility.

The pre‐ovulatory luteinizing hormone surge is followed by down‐regulation of CYP19A1, HSD3B1, and CYP17A1 and chromatin condensation of the corresponding promoters in bovine follicles

The pre-ovulatory luteinizing hormone (LH) surge induces an extensive molecular, physiological, and morphological reorganization of the bovine follicle. This study was designed to elucidate if chromatin modulation is involved in the LH-induced gene regulation. Granulosa and theca of well-characterized large bovine follicles were isolated before and after the LH surge. CYP19A1, HSD3B1, and CYP17A1 transcripts, which encode key enzymes of steroid hormone biosynthesis, were quantified by real-time PCR (qPCR) and the degree of chromatin condensation was determined by DNase I protection assays. After LH, granulosa-specific CYP19A1 and theca-specific CYP17A1 transcripts were almost completely down-regulated. Also, the abundance of HSD3B1 transcripts was reduced. The promoter chromatin of HSD3B1 and particularly of CYP19A1 was significantly less accessible to DNAse I in both cell types after LH, whereas the chromatin accessibility of the CYP17A1 promoter changed only in the theca. Correlation analysis revealed partly, highly significant negative correlations between transcript abundance and protection from DNase I digestion of the corresponding chromatin. The data strongly suggest that LH induces cell type- and gene-specific chromatin condensation in the pre-ovulatory bovine follicle. This epigenetic mechanism might be involved in the pre-ovulatory down-regulation of genes.

Abstract 2941: Rapid changes in chromatin structure in response to extracellular signals

Abstract Immediate early genes (IEGs) are well known to be rapidly and transiently induced at high levels of expression in response to various extracellular signals such as growth factors. These rapid changes in the transcriptional state of this class of genes raised questions regarding the role of higher order chromatin structure in this transient event of inducible gene expression. In these studies, we compared the degree of chromatin condensation in Jurkat cells before and after mitogen stimulation of c-fos, a well-known IEG. Soluble chromatin obtained from unstimulated and stimulated cells were collected for a sucrose gradient sedimentation assay in order to separate chromatin fibers by means of size and more importantly by the extent of structural condensation of the chromatin. Real time-PCR analysis of c-fos was then used as a tool to quantify the distribution of open and closed chromatin fibers across the collected fractions obtained from the sucrose gradients. The results suggested a major decondensation of the chromatin fiber at the c-fos gene upon stimulation. The unfolding of the chromatin structure upon stimulation is paired with a dramatic increase in histone acetylation as detected by chromatin immunoprecipiation (ChIP) at the promoter region. Histone hyperacetylation is known to cause destabilization of the higher order chromatin structure leading to an increase in chromatin accessibility as clearly shown in our DNase hypersensitivity studies. Taken together, we have detected a rapid decondensation of the chromatin fiber, which is believed to facilitate the rapid transcriptional response to extracellular signals. Our current interest lies in obtaining further information regarding the molecular events that are more directly related to destabilization of the higher order chromatin structure upon mitogen stimulation. Testing of various molecular candidates involved in chromatin destabilizing events include phosphorylation of linker histone H1, recruitment of histone variants and dramatic histone modications such as ADP-polyribosylation at the c-fos promoter region. A more detailed understanding on the rapid decondensation of the chromatin fiber would provide valuable information regarding the cascade of events that must take place in response to stress or extracellular signals. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2941.

Chromatin Configurations in the Ferret Germinal Vesicle that Reflect Developmental Competence for In Vitro Maturation

In several mammalian species, the configuration of germinal vesicle (GV) chromatin correlates with the developmental competence of oocytes. Yet, no study has been published on the configuration of GV chromatin in ferret, nor is it known whether a specific configuration predicts meiotic competence in this species, in spite of the potential importance of ferret cloning to the study of human disease and to species conservation efforts. Here, we report on an analysis of the chromatin configuration in ferret GV oocytes and on how they correlate with meiotic development. Three distinct configurations were identified based on the degree of chromatin condensation: (1) fibrillar chromatin (FC), featuring strands of intertwined chromatin occupying most of the visible GV region; (2) intermediate condensed chromatin (ICC), characterized by dense, irregular chromatin masses throughout the GV; and (3) condensed chromatin (CC), which is highly compact and centered around the nucleolus. We also found that chromatin configuration was related to the extent of association with cumulus cells in cumulus-oocyte complexes; CC-configured oocytes were most often surrounded by a compact cumulus layer and also a compact corona but FC-configured oocytes were associated with neither. In addition, increasing chromatin condensation corresponded to an increase in oocyte diameter. Finally, following in vitro culture, significantly more CC-configured oocytes underwent maturation to meiotic metaphase II than did FC- or ICC-configured oocytes. We conclude that, in ferret, chromatin condensation is related to the sequential achievement of meiotic competencies during oocyte growth and differentiation, and thus can be used as a predictor of competence.

Quantitative PCR analysis of diepoxybutane and epihalohydrin damage to nuclear versus mitochondrial DNA

The bifunctional alkylating agents diepoxybutane (DEB) and epichlorohydrin (ECH) are linked to the elevated incidence of certain cancers among workers in the synthetic polymer industry. Both compounds form interstrand cross-links within duplex DNA, an activity suggested to contribute to their cytotoxicity. To assess the DNA targeting of these compounds in vivo, we assayed for damage within chicken erythro-progenitor cells at three different sites: one within mitochondrial DNA, one within expressed nuclear DNA, and one within unexpressed nuclear DNA. We determined the degree of damage at each site via a quantitative polymerase chain reaction, which compares amplification of control, untreated DNA to that from cells exposed to the agent in question. We found that ECH and the related compound epibromohydrin preferentially target nuclear DNA relative to mitochondrial DNA, whereas DEB reacts similarly with the two genomes. Decreased reactivity of the mitochondrial genome could contribute to the reduced apoptotic potential of ECH relative to DEB. Additionally, formation of lesions by all agents occurred at comparable levels for unexpressed and expressed nuclear loci, suggesting that alkylation is unaffected by the degree of chromatin condensation.

108 CALYCULIN-A INDUCES CHROMOSOME CONDENSATION FOR CYTOGENETIC ANALYSIS IN BOVINE AND MURINE BLASTOMERES

Cytogenetic studies of preimplantation embryos have traditionally used interphase fluorescence in situ hybridization (FISH) to examine chromosome copy number, chromosomal rearrangements, or sex determination. Comprehensive analysis of chromosomes is hindered by the low mitotic index of blastomeres, combined with the technical limitations of interphase FISH. Efforts to overcome these limitations include inducing premature chromosome condensation (PCC) for metaphase FISH, by fusing blastomeres with metaphase II-stage oocytes, or with time-consuming incubations with okadaic acid or vinblastine. Our objective was to evaluate Calyculin-A (CA) as an alternative to induce PCC in blastomeres. In vitro-produced bovine 8-cell embryos and frozen–thawed mouse 8-cell embryos were cultured with CA and examined for changes in morphology before fixation. Colcemid (0.1 mg mL–1), the standard for cytogenetic analysis, and vehicle served as positive and negative controls. Blastomeres were washed in hypotonic solution, loaded onto slides, and fixed in methanol:acetic acid (3:1). The degree of chromatin condensation and quality of chromosome spreads were determined by 42,6-diamidino-2-phenylindole staining and visualization on an epifluorescence microscope (100�). Experiment 1 (1 rep, 136 cells) tested dose of CA on bovine blastomeres at 50, 100, 150, 250, 500, and 750 nm. Experiments 2 and 3 tested culture duration of CA (50 nm) at 60, 120, and 180 min in bovine blastomeres and 60, 90, and 120 min in mouse blastomeres (2 reps each, 132 and 207 cells, respectively). Data were analyzed by chi-square with significance deemed P &lt; 0.05. Cell lysis and blebbing was observed in bovine blastomeres treated with greater than 150 nm CA. Duration of CA treatment affected the frequency of bovine and mouse blastomeres undergoing PCC. Fewer bovine blastomeres treated for 60 min underwent chromatin condensation compared to blastomeres treated for at least 120 min with 50 nm CA (25% v. 100%; P &lt; 0.05). In mice, the frequency of blastomeres undergoing PCC was lower (43%) for 50 nm CA at 60 min than at 90 and 120 min (90 and 100%, respectively; P &lt; 0.05). Chromatin condensation suitable for cytogenetic analysis was 20, 34, and 20%, respectively, but did not differ (P &gt; 0.10). Further examination (Exp. 4, 5 reps, 293 cells) comparing the degree of condensation revealed 55% of the total bovine blastomeres treated with 50 nm CA for 120 min were suitable for cytogenetic analysis, as compared to 30% treated with colcemid for 16 h (P &lt; 0.05). Bovine and mouse blastomeres treated with vehicle had lower PCC than all other treatments averaging 2 and 7%, respectively (P &lt; 0.05). These results suggest that CA can rapidly induce PCC in blastomeres from pre-implantation bovine and murine embryos, but the degree of chromatin condensation may not always be suitable for detailed cytogenetic analysis from a single blastomere.