CpG DNA methylation changes during epididymal sperm maturation in bulls.

BACKGROUND: During epididymal transit spermatozoa acquire specific morphological features which enhance their ability to swim in a progressive manner and interact with the oocytes. At the same time, sperm cells undergo specific molecular rearrangements essential for the fertilizing sperm to drive a correct embryo development. To assess epigenetic sperm changes during epididymal maturation, the caput, corpus and cauda epididymis sperm tracts were isolated from eight bulls and characterized for different sperm quality parameters and for CpG DNA methylation using Reduced Representation Bisulfite Sequencing (RRBS) able to identify differentially methylated regions (DMRs) in higher CpG density regions. RESULTS: Caput sperm showed significant variation in motility and sperm kinetics variables, whereas spermatozoa collected from the corpus presented morphology variation and significant alterations in variables related to acrosome integrity. A total of 57,583 methylated regions were identified across the eight bulls, showing a significantly diverse distribution for sperm collected in the three epididymal regions. Differential methylation was observed between caput vs corpus (n = 11,434), corpus vs cauda (n = 12,372) and caput vs cauda (n = 2790). During epididymal transit a high proportion of the epigenome was remodeled, showing several regions in which methylation decreases from caput to corpus and increases from corpus to cauda. CONCLUSIONS: Specific CpG DNA methylation changes in sperm isolated from the caput, corpus, and cauda epididymis tracts are likely to refine the sperm epigenome during sperm maturation, potentially impacting sperm fertilization ability and spatial organization of the genome during early embryo development.

Comparison between indicine and taurine cattle DNA methylation reveals epigenetic variation associated to differences in morphological adaptive traits.

Indicine and taurine subspecies present distinct morphological traits as a consequence of environmental adaptation and artificial selection. Although the two subspecies have been characterized and compared at genome-wide level and at specific loci, their epigenetic diversity has not yet been explored. In this work, Reduced Representation Bisulphite Sequencing (RRBS) profiling of the taurine Angus (A) and indicine Nellore (N) cattle breeds was applied to identify methylation differences between the two subspecies. Genotyping by sequencing (GBS) of the same animals was performed to detect single nucleotide polymorphisms (SNPs) at cytosines in CpG dinucleotides and remove them from the differential methylation analysis. A total of 660,845 methylated cytosines were identified within the CpG context (CpGs) across the 10 animals sequenced (5 N and 5 A). A total of 25,765 of these were differentially methylated (DMCs). Most DMCs clustered in CpG stretches nearby genes involved in cellular and anatomical structure morphogenesis. Also, sequences flanking DMC were enriched in SNPs compared to all other CpGs, either methylated or unmethylated in the two subspecies. Our data suggest a contribution of epigenetics to the regulation and divergence of anatomical morphogenesis in the two subspecies relevant for cattle evolution and sub-species differentiation and adaptation.

Small RNA sequencing of cryopreserved semen from single bull revealed altered miRNAs and piRNAs expression between High- and Low-motile sperm populations.

BACKGROUND: Small RNAs present in bovine ejaculate can be linked to sperm abnormalities and fertility disorders. At present, quality parameters routinely used in semen evaluation are not fully reliable to predict bull fertility. In order to provide additional quality measurements for cryopreserved semen used for breeding, a method based on deep sequencing of sperm microRNA (miRNA) and Piwi-interacting RNA (piRNA) from individual bulls was developed. To validate our method, two populations of spermatozoa isolated from high and low motile fractions separated by Percoll were sequenced, and their small RNAs content characterized. RESULTS: Sperm cells from frozen thawed semen samples of 4 bulls were successfully separated in two fractions. We identified 83 miRNAs and 79 putative piRNAs clusters that were differentially expressed in both fractions. Gene pathways targeted by 40 known differentially expressed miRNAs were related to apoptosis. Dysregulation of miR-17-5p, miR-26a-5p, miR-486-5p, miR-122-5p, miR-184 and miR-20a-5p was found to target three pathways (PTEN, PI3K/AKT and STAT). CONCLUSIONS: Small RNAs sequencing data obtained from single bulls are consistent with previous findings. Specific miRNAs are differentially represented in low versus high motile sperm, suggesting an alteration of cell functions and increased germ cell apoptosis in the low motile fraction.

Expression profiling of genes involved in the formation of aroma in two peach genotypes.

The expression profile of flavour-related genes during ripening was investigated in two peach genotypes, Bolero and OroA, which have been selected for their contrasting aroma/ripening behaviour. A new peach microarray containing 4776 oligonucleotide probes corresponding to a set of ESTs specifically enriched in secondary metabolism (µPEACH2.0) was designed to investigate transcriptome changes during three fruit ripening stages, revealing 1807 transcripts differentially expressed within and between the two genotypes. Differences in the expression of genes involved in the biosynthesis of aroma compounds were detected during the ripening process within and between the two genotypes. In particular, a subset of 12 transcripts involved in metabolism of esters, norisoprenoids, phenylpropanoids and lactones, varied in expression during ripening and between Bolero and OroA.

An alternative method for noise analysis using pixel variance as part of quality control procedures on digital mammography systems.

According to the European Guidelines for quality assured breast cancer screening and diagnosis, noise analysis is one of the measurements that needs to be performed as part of quality control procedures on digital mammography systems. However, the method recommended in the European Guidelines does not discriminate sufficiently between systems with and without additional noise besides quantum noise. This paper attempts to give an alternative and relatively simple method for noise analysis which can divide noise into electronic noise, structured noise and quantum noise. Quantum noise needs to be the dominant noise source in clinical images for optimal performance of a digital mammography system, and therefore the amount of electronic and structured noise should be minimal. For several digital mammography systems, the noise was separated into components based on the measured pixel value, standard deviation (SD) of the image and the detector entrance dose. The results showed that differences between systems exist. Our findings confirm that the proposed method is able to discriminate systems based on their noise performance and is able to detect possible quality problems. Therefore, we suggest to replace the current method for noise analysis as described in the European Guidelines by the alternative method described in this paper.

Avian TAP genes: detection of nucleotide polymorphisms and comparative analysis across species.

TAP1 and TAP2 genes code for the two subunits of the transporter associated with antigen processing (TAP), and in chicken they are located between the two MHC class I genes. Using primers based on chicken sequences, the genomic regions corresponding to chicken TAP1 exons 6 to 7 and TAP2 exons 4 to 6 (which encode portions of the chicken TAP1 and TAP2 molecules corresponding to the human peptide-binding regions) were amplified and sequenced from chicken (70 birds), turkey (24), pheasant (6), and guinea fowl (7). A total of 80 within-species single nucleotide polymorphisms (SNPs) were identified. None of the chicken SNPs detected here was present in public databases. The SNP frequencies in chicken were 9.57 SNP/kb in TAP1 and 19.16 SNP/kb in TAP2, while turkey showed similar SNP frequencies in the two genes. Putative amino acid sequences were inferred to identify non-synonymous substitutions. The alignment of the consensus polypeptide sequences showed that most of the amino acid variations were conserved or semi-conserved substitutions. In conclusion, a high variability in the level of nucleotide polymorphism was observed within the two genes, with chicken showing the highest polymorphism rate in both genes. Most of the SNPs identified were within introns, and a general conservation of both amino acid numbers and characteristics of residues among and within the species was found. These data underline the functional importance of these molecules, but also suggest their capacity to bind different antigenic peptides.

Physical characteristics of five clinical systems for digital mammography.

The purpose of this study was to evaluate and compare the physical characteristics of five clinical systems for digital mammography (GE Senographe 2000D, Lorad Selenia M-IV, Fischer Senoscan, Agfa DM 1000, and IMS Giotto) currently in clinical use. The basic performances of the mammography systems tested were assessed on the basis of response curve, modulation transfer function (MTF), noise power spectrum, noise equivalent quanta (NEQ), and detective quantum efficiency (DQE) in an experimental setting closely resembling the clinical one. As expected, all the full field digital mammography systems show a linear response curve over a dynamic range from 3.5 to 500 microGy (0.998

Single nucleotide polymorphism discovery in the avian Tapasin gene.

Tapasin is a transmembrane glycoprotein located in the endoplasmic reticulum. Its function is to assist the assembly of major histocompatibility complex class I molecules. The chicken Tapasin gene includes 8 exons and is localized inside the major histocompatibility complex between the 2 class IIbeta genes. The aim of the current study was the estimation of single nucleotide polymorphism frequency within the avian Tapasin gene. The Tapasin gene sequence from exon 5 to exon 6 was amplified for the chicken, turkey, and pheasant, and sequences of different lengths were obtained. The sequence analysis based on PolyBayes identified 25 putative single nucleotide polymorphism sites when the 3 species were compared. The coding sequences were further translated and analyzed to identify amino acid substitutions. The results indicated that polymorphisms within this region of the gene was mainly observed in the heterozygous state. The level of conservation of the Tapasin gene sequence among species is likely to be related to the functional importance of the gene.

Use of TLD in evaluating diagnostic reference levels for some radiological examinations.

The European Council Directive 97/43 introduces diagnostic reference levels (DRL) for all medical examinations involving ionising radiation. Each department has to evaluate patient dose and to compare that value with the DRL adopted by its member state. Italian law, applying the Directive, states that reference levels must be measured every 2 years. Quantities that must be measured are entrance surface dose or air kerma, or other dosimetric quantities. In our work, clinical measurements on patients were made by positioning a thermoluminescence dosemeter (TLD) over the skin of a statistically significant number of patients for each projection of each examination. As there is no national guideline for these measurements in Italy, the aim of this work was to establish a method based both on European publications and on clinical experience. Three different modalities were considered: conventional radiography, computed radiography and mammography. Accordingly, differently shaped types of TLD were required, especially for mammography where the beam energy is lower.

Specific combinations of zein genes and genetic backgrounds influence the transcription of the heavy-chain zein genes in maize opaque-2 endosperms.

The transcript levels of heavy-chain zein genes (zH1 and zH2) and the occurrence of the zH polypeptides in different opaque-2 (o2) lines were investigated by RNA-blot analyses and by sodium dodecylsulfate-polyacrylamide gel electrophoresis or two-dimensional gel electrophoresis protein fractionations. Four mutant alleles o2R, o2T, o2It, and o2-676 introgressed into different genetic backgrounds (GBs) were considered. The mono-dimensional gel electrophoresis zein pattern can be either conserved or different among the various GBs carrying the same o2 allele. Likewise, in the identical GB carrying different o2 alleles, the zein pattern can be either conserved or differentially affected by the different mutant allele. Zein protein analysis of reciprocal crosses between lines with different o2 alleles or the same o2 showed in some case a more than additive zH pattern in respect to the o2 parent lines. Electrophoretic mobility shift assay approaches, with O2-binding oligonucleotide and endosperm extracts from the above o2 lines, failed to reveal o2-specific retarded band in any of the o2 extracts. The results suggest that the promoter of some zH1 and zH2 contains motif(s) that can respond to factors other than O2.

Phosphorylation of Opaque2 changes diurnally and impacts its DNA binding activity.

In the maize endosperm, the Opaque2 (O2) basic leucine zipper transcriptional activator regulates the expression of a subset of the zein seed storage protein gene family. Immunodetection of wild-type or mutant O2 polypeptides fractionated by SDS-PAGE resolved a closely spaced doublet migrating in the 68- to 72-kD range, whereas by using isoelectric focusing, seven to nine isoforms were detected for each allele. Phosphatase treatment simplified the protein patterns to a single band corresponding to the nonphosphorylated component. In vivo and in vitro labeling confirmed that O2 can be phosphorylated. In protein gel blots probed with DNA, only the nonphosphorylated and hypophosphorylated O2 polypeptides were able to bind an oligonucleotide containing the O2 binding sequence. Upon in situ dephosphorylation of the focused isoforms by phosphatase treatment of the isoelectric focusing filter, the hyperphosphorylated forms acquired DNA binding activity. The ratio among the various isoforms remained constant throughout the developmental stages of endosperm growth but changed from daytime to nighttime, with a significant increase of the hyperphosphorylated forms during the night period. These results indicate that O2 exists in vivo as a pool of differently phosphorylated polypeptides and demonstrate that O2 DNA binding activity is modulated by a phosphorylation/dephosphorylation mechanism that appears to be influenced by environmental conditions.

The nucleotide sequence of a CpG island demonstrates the presence of the first exon of the gene encoding the human lysosomal membrane protein lamp2 and assigns the gene to Xq24.

An EagI-EcoRI clone of human genomic DNA, p2-7, mapped to Xq24 has been sequenced. This analysis has confirmed the presence of a CpG island and has identified the first exon of the human LAMP2 gene, encoding a glycoprotein of the lysosomal membrane. Since the p2-7 clone corresponds to single-copy DNA, we can assign the human LAMP2 gene to Xq24.