Spermatozoal sensitive biomarkers to defective protaminosis and fragmented DNA

Human sperm DNA damage may have adverse effects on reproductive outcome. Infertile men possess substantially more spermatozoa with damaged DNA compared to fertile donors. Although the extent of this abnormality is closely related to sperm function, the underlying etiology of ensuing male infertility is still largely controversial. Both intra-testicular and post-testicular events have been postulated and different mechanisms have been proposed to explain the presence of damaged DNA in human spermatozoa. Three among them, i.e. abnormal chromatin packaging, oxidative stress and apoptosis, are the most studied and discussed in the present review. Furthermore, results from numerous investigations are presented, including our own findings on these pathological conditions, as well as the techniques applied for their evaluation. The crucial points of each methodology on the successful detection of DNA damage and their validity on the appraisal of infertile patients are also discussed. Along with the conventional parameters examined in the standard semen analysis, evaluation of damaged sperm DNA seems to complement the investigation of factors affecting male fertility and may prove an efficient diagnostic tool in the prediction of pregnancy outcome

Chromatin and epigenetics: current biophysical views

Recent advances in high-throughput sequencing experiments and their theoretical descriptions have determined fast dynamics of the "chromatin and epigenetics" field, with new concepts appearing at high rate. This field includes but is not limited to the study of DNA-protein-RNA interactions, chromatin packing properties at different scales, regulation of gene expression and protein trafficking in the cell nucleus, binding site search in the crowded chromatin environment and modulation of physical interactions by covalent chemical modifications of the binding partners. The current special issue does not pretend for the full coverage of the field, but it rather aims to capture its development and provide a snapshot of the most recent concepts and approaches. Eighteen open-access articles comprising this issue provide a delicate balance between current theoretical and experimental biophysical approaches to uncover chromatin structure and understand epigenetic regulation, allowing free flow of new ideas and preliminary results

Chromosomal localization of a highly repeated EcoRI DNA fragment in Megoura viciae (Homoptera, Aphididae) by nick translation and fluorescence in situ hybridization.

To investigate the genome of the aphid Megoura viciae at molecular level, we have studied total DNA by agarose gel electrophoresis after cleavage with different restriction endonucleases. EcoRI digestion produced a highly repeated DNA fragment, about 600 pb long. The contribution of this EcoRI element to the total genome of M. viciae was estimated at about 6% by means of densitometric scanning of agarose gel photographs. The chromosomal localization of this fragment, investigated by fluorescent in situ hybridization (FISH), constantly showed one large and two narrower fluorescent bands located on the X chromosome, all corresponding to C-positive heterochromatic areas. These results are in full accordance with the data obtained by in situ nick translation experiments carried out after EcoRI digestion, and clearly demonstrate that a substantial amount of M. viciae heterochromatin consists of EcoRI fragments which are mainly located on the X chromosome. Using the EcoRI restriction fragment as a molecular probe may be a practical tool for the investigation of taxonomic and evolutionary relationships in this group of insects

Heterochromatin heterogeneity in the holocentric X chromatin of Megoura viciae (Homoptera, Aphididae).

Holocentric chromosomes, prepared by spreading embryo cells obtained from Megoura viciae parthenogenetic females, have been C-banded, enzymatically digested in situ using the specific endonucleases DdeI (C↓TNAG), DraI (TTT↓AAA), Tru9I (TT↓AA), and CfoI (GCG↓C), and subsequently stained with Giemsa, DAPI, CMA3, and AgNO3. We observed that the X chromosome had the best defined banding patterns. In the M. viciae X chromosome there is a certain amount of heterogeneity in heterochromatic DNA composition. In fact, the GC-rich NOR-associated heterochromatin differs from other heterochromatic bands that are characterized by AT-rich DNAs. Our data also indicate that, in M. viciae holocentric chromosomes, all heterochromatic blocks are accessible to in situ enzyme attack, the only limit to the digestion being the presence or absence of recognition targets. This is an interesting point, since, in monocentric chromosomes, it is well known that in situ endonuclease digestion is heavily affected not only by DNA base composition but also by chromatin compactness that may limit enzyme accessibility to their specific targets. Key words : heterochromatin, holocentric chromosomes, aphids

Cytological and electrophoretic analysis of DNA methylation in the holocentric chromosomes of Megoura viciae (Homoptera, Aphididae).

Chromosomal and purified DNA methylation patterns were determined in the holocentric chromosomes of Megoura viciae by treatment with MspI and HpaII. Both enzymes produced a clear C-like banding pattern but widely digested one telomere of the X chromosome, which appeared as heterochromatic after C-banding treatment and brightly fluorescent after chromomycin A3 staining. Quantitative microfluorometric evaluations of DNA extraction performed on cytological preparations showed that both isoschizomers resulted in the same DNA extraction (about 30%). Contrary to what was found by in situ endonuclease treatment, the electrophoretic patterns of purified and digested DNA showed that digestion with MspI was slightly more extensive than that with HpaII in a zone of fragments ranging from 23 to 9 kb. This result indicates that aphid chromatin is not wholly unmethylated. The discrepancy between electrophoretic and cytological data has been explained by taking into consideration that DNA fragments with high molecular weights could be cleaved in situ by the enzymes but not extracted from the chromatin

Heterochromatin heterogeneity in the holocentric X chromatin of Megoura viciae (Homoptera, Aphididae).

Holocentric chromosomes, prepared by spreading embryo cells obtained from Megoura viciae parthenogenetic females, have been C-banded, enzymatically digested in situ using the specific endonucleases DdeI (C↓TNAG), DraI (TTT↓AAA), Tru9I (TT↓AA), and CfoI (GCG↓C), and subsequently stained with Giemsa, DAPI, CMA3, and AgNO3. We observed that the X chromosome had the best defined banding patterns. In the M. viciae X chromosome there is a certain amount of heterogeneity in heterochromatic DNA composition. In fact, the GC-rich NOR-associated heterochromatin differs from other heterochromatic bands that are characterized by AT-rich DNAs. Our data also indicate that, in M. viciae holocentric chromosomes, all heterochromatic blocks are accessible to in situ enzyme attack, the only limit to the digestion being the presence or absence of recognition targets. This is an interesting point, since, in monocentric chromosomes, it is well known that in situ endonuclease digestion is heavily affected not only by DNA base composition but also by chromatin compactness that may limit enzyme accessibility to their specific targets. Key words : heterochromatin, holocentric chromosomes, aphids