ABSTRACT
Background: There is an association between aging ana an increased number ofsperms with alterations in nuclear DNA. Aim: To study the association between age and fragmentation of sperm DNA. Material andMethods: Sixty two volunteers provided semen for analysis. These were separated in a group aged less than forty years and a second group aged more than forty years. Sperm DNA fragmentation was studied by TÚNEL (terminal deoxynucleotidyl transferase-mediated2'-deoxyuridine 5'-triphosphate nick end-labeling) and SCD (sperm chromatin dispersión test) assays. Results: Compared with theiryounger counterparts, patients aged more than 40years had a higher proportion ofsperms with DNA fragmentation by TÚNEL (20 ±1.3 and24 ± 1.9 percent respectively, p < 0.05) and SCD (22 ± 1.4 and26 ± 1.6 percent respectively, p < 0.05). The results ofboth assays had a correlation coefficientofO.8. No differences between groups were observed for other seminal parameters. Conclusions: Sperm DNA fragmentation increases with age in males.
Subject(s)
Adolescent , Adult , Humans , Male , Middle Aged , Young Adult , Chromatin/chemistry , DNA Fragmentation , In Situ Nick-End Labeling/methods , Semen Analysis/methods , Spermatozoa/ultrastructure , Age Factors , Sperm Count , Sperm Motility , Spermatozoa/physiologyABSTRACT
We used a rapid and simple protocol using lysolecithin for mapping HS sites in vivo. The protocol is based on partial digestion with DNase I of exponentially growing cells following permeabilization by short treatment with lysolecithin. Using this protocol, we analyzed the chromatin structure of the region surrounding two overlapping elements, an origin of bidirectional DNA replication and the GAS41 promoter, in chicken myelomonocytic HD11 cells arrested in G0, G0 and S phases as well as at the G0/S border. The results show that the chromatin of this region became more nuclease sensitive when cells were arrested in G0 phase and that this change in chromatin structure was reversible after the cells began to enter S phase.
Subject(s)
Humans , Cell Cycle/genetics , Chromatin/genetics , DNA Replication/genetics , Transcription Factors/genetics , Cell Line , Cell Cycle/physiology , Chromatin/chemistry , Chromatin/metabolismABSTRACT
The ability to detect nuclear damage is an important tool for the development of sperm preservation methods. We used the acridine orange test (AOT) and the terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling (TUNEL) assay to assess the DNA status of sperm cells preserved with different lyophilization media. The AOT did not detect any differences between different lyophilization media. However, differences in DNA integrity were observed among treatments with the TUNEL assay, suggesting that TUNEL is a more sensitive method to evaluate sperm DNA. The use of TCM 199 and 10% FCS as a lyophilization medium resulted in 14% of the cells with DNA fragmentation in TUNEL test. The AOT indicated only 4% of the cells with chromatin damage, with this same treatment, with no significant differences when compared to the other treatments. The degree of DNA fragmentation was negatively related to fertilizing potential, as sperm DNA damage was inversely correlated with pro-nucleus formation. The TUNEL assay was found to be an efficient method to detect DNA damage in sperm, and it could be used as a tool to predict male fertility
Subject(s)
Animals , Male , Cattle , DNA , Chromatin/chemistry , Coloring Agents , Flow Cytometry , Spermatozoa/metabolism , DNA , Chromatin/metabolism , DNA Fragmentation , In Situ Nick-End Labeling , Nucleic Acid Conformation , Protein Conformation , Staining and LabelingABSTRACT
Mithramycin (MTR) is an anti-cancer antibiotic that blocks the macromolecular biosynthesis via reversible interaction with DNA template in the presence of bivalent metal ion such as Mg2+. In absence of DNA, mithramycin forms two types of complexes with Mg2+, complex I (with 1:1 stoichiometry in terms of MTR: Mg2+) and complex II (with 1:2 stoichiometry in terms of MTR: Mg2+). In an eukaryotic system, the drug would interact with chromatin, a protein-DNA complex. We have employed the spectroscopic techniques such as absorption and fluorescence to study the interaction of MTR: Mg2+ complexes with rat liver chromatin. In this report, we have shown that the two types of ligands have different binding potentials with the same chromatin. This supports our proposition that complexes I and II, are different molecular species. We have also shown that the histone protein(s) reduce the binding potential and the number of available sites for both ligands.
Subject(s)
Animals , Chromatin/chemistry , Dose-Response Relationship, Drug , Histones/chemistry , Kinetics , Ligands , Liver/metabolism , Magnesium/metabolism , Male , Nucleic Acid Synthesis Inhibitors/chemistry , Plicamycin/chemistry , Protein Binding , Rats , Spectrometry, FluorescenceABSTRACT
The critical electrolyte concentrations (CEC) of sperm chromatin from animal species known or suspected to contain histone H1 variants were compared by examining the affinity of their DNA-protein complexes for toluidine blue in the presence of Mg2+. Bullfrog, sea urchin, bee and bumblebee spermatozoa were studied. The CEC for Rana catesbeiana and two sea urchin species were similar to that of histone H5-containing chromatin from chicken erythrocytes, thus confirming the biochemical and structural similarities of these DNA-protein complexes. The CEC for bees and the bumblebee, Bombus atratus, showed no particular phylogenetic relationship. We concluded that the CEC of histone H1-containing sperm cell chromatin is a useful indicator of variability in DNA-protein complexes but is of little phylogenetic value.
Subject(s)
Animals , Chromatin/chemistry , Electrolytes/analysis , Histones/analysis , Spermatozoa/cytology , Bees , DNA , Hymenoptera , Rana catesbeiana , Sea UrchinsABSTRACT
A Concentraçao Crítica de eletrólitos, na citoquímica e histoquímica tradicionais, tem se mostrado de grande importância para o estudo de grupamentos aniônicos na célula e no meio extracelular. Por esta técnica identificam-se compostos aniônicos da matriz extracelular (proteoglicanos e glicosaminoglicanos) e da cromatina e citoplasma (ácidos nucléicos). Ela baseia-se na competiçao de sais inorgânicos com corantes tiazínicos. Este trabalho tem como objetivo fazer uma abordagem técnico-metodológico desta técnica, principalmente com o intuito de divulgar, uma técnica de fácil exequibilidade e custo baixo para os laboratórios de histotecnologia e histopatologia.
Subject(s)
Electrolytes/analysis , Extracellular Matrix/chemistry , Chromatin/chemistrySubject(s)
Humans , DNA Damage/genetics , DNA/ultrastructure , Oxidants/adverse effects , Chromatin/chemistry , Chromatin/enzymology , DNA Damage , DNA, Mitochondrial/chemistry , DNA, Mitochondrial/history , DNA, Mitochondrial/ultrastructure , DNA, Viral/chemistry , Histones/chemistry , Histones/drug effects , PedigreeABSTRACT
The organization of chromatin in protists presents some characteristic features. In Trypanosoma cruzi, no condensation of chromatin into chromosomes is observed during cell division. A systematic characterization of histones should provide information on this peculiar behaviour. Histone H2B from this parasite was characterized by selective dissociation from chromatin in 0.8 M NaCl, by its elution pattern in narrow-bore reversed phase high performance liquid chromatography, by polyacrylamide gel electrophoresis and by partial sequencing of its amino terminal domain. This chromosomal protein differs from histone H2B of other species. The first 12 amino acids are missing which explains its lower molecular weight when compared to human histone H2B. Correspondingly, the amino terminal domain of T. cruzi histone H2B is 25-30 percent shorter than other histones H2B. Moreover, three out of four acetylation sites present in human histone H2B are missing in T. cruzi histone H2B. The differences in size and in acceptor sites for acetylation of T. cruzi histone H2B when compared to human histone H2B may represent a functional feature to consider for the understanding of the chromatin cycle of condensation in this parasite
Subject(s)
Animals , Humans , Chromatin/chemistry , Histones/isolation & purification , Protozoan Proteins/isolation & purification , Trypanosoma cruzi/chemistry , Amino Acid Sequence , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Molecular Sequence Data , Sequence Alignment , Sodium ChlorideABSTRACT
To assess the possible functional role of single-strand DNA-binding (SSB) proteins in eucaryotic cell, a comparative study was made of SSB-proteins isolated from chromatin and the nonchromatin fractions of Ehrlich ascites tumour (EAT) cells. No appreciable differences between the two groups could be found either in SDS-gel electrophoretic patterns or in the ssDNA-binding capacity and stimulation of DNA replication in permeable EAT cells. However, the chromatin SSB-proteins incorporated 1.4-times more labelled phosphate in vivo; phosphate assays in the isolated chromatin and nonchromatin SSB-proteins yielded ca. 3 and 2 moles Pi/mole protein, respectively. Both preparations could be further phosphorylated in vitro with Ca-phospholipid-dependent protein kinase and the catalytic subunit of cAMP-dependent protein kinase, but the non-chromatin proteins were phosphorylated to a greater degree. In parallel with phosphorylation, the SSB-proteins displayed stronger binding to ssDNA cellulose. Phosphorylation may thus be a means of regulating the functions of SSB-proteins, in particular their interaction with chromatin.