Increased DNA strand breaks in spermatozoa of Pxt1 knockout mice.

CONTEXT: The Pxt1 gene encodes a male germ cell-specific protein and its overexpression results in male germ cell degeneration and male infertility in transgenic mice. AIMS: The analysis of the function of Pxt1 during mouse spermatogenesis. METHODS: The phenotype of Pxt1 knockout mice was characterised by testicular histology, assessment of semen parameters including sperm motility, and DNA fragmentation by flow cytometry. Gene expression was analysed using RT-PCR. Fertility of mutants was checked by standard breeding and competition breeding tests. KEY RESULTS: In Pxt1 -/- mice, a strong increase in the sperm DNA fragmentation index (DFI) was observed, while other sperm parameters were comparable to those of control animals. Despite enhanced DFI, mutants were fertile and able to mate in competition with wild type males. CONCLUSIONS: Pxt1 induces cell death; thus, the higher sperm DFI of mice with targeted deletion of Pxt1 suggests some function for this gene in the elimination of male germ cells with chromatin damage. IMPLICATIONS: Ablation of mouse Pxt1 results in enhanced DFI. In humans, the homologous PXT1 gene shares 74% similarity with the mouse gene; thus, it can be considered a candidate for mutation screening in patients with increased DFI.

Role of Non-Coding Regulatory Elements in the Control of GR-Dependent Gene Expression.

The glucocorticoid receptor (GR, also known as NR3C1) coordinates molecular responses to stress. It is a potent transcription activator and repressor that influences hundreds of genes. Enhancers are non-coding DNA regions outside of the core promoters that increase transcriptional activity via long-distance interactions. Active GR binds to pre-existing enhancer sites and recruits further factors, including EP300, a known transcriptional coactivator. However, it is not known how the timing of GR-binding-induced enhancer remodeling relates to transcriptional changes. Here we analyze data from the ENCODE project that provides ChIP-Seq and RNA-Seq data at distinct time points after dexamethasone exposure of human A549 epithelial-like cell line. This study aimed to investigate the temporal interplay between GR binding, enhancer remodeling, and gene expression. By investigating a single distal GR-binding site for each differentially upregulated gene, we show that transcriptional changes follow GR binding, and that the largest enhancer remodeling coincides in time with the highest gene expression changes. A detailed analysis of the time course showed that for upregulated genes, enhancer activation persists after gene expression changes settle. Moreover, genes with the largest change in EP300 binding showed the highest expression dynamics before the peak of EP300 recruitment. Overall, our results show that enhancer remodeling may not directly be driving gene expression dynamics but rather be a consequence of expression activation.

Influence of a low dose of silver nanoparticles on cerebral myelin and behavior of adult rats.

Nanoscale particles have large surface to volume ratio that significantly enhances their chemical and biological reactivity. Although general toxicity of nano silver (nanoAg) has been intensively studied in both in vitro and in vivo models, its neurotoxic effects are poorly known, especially those of low-dose exposure. In the present study we assess whether oral administration of nanoAg influences behavior of exposed rats and induces changes in cerebral myelin. We examine the effect of prolonged exposure of adult rats to small (10nm) citrate-stabilized nanoAg particles at a low dose of 0.2mg/kg b.w. (as opposed to the ionic silver) in a comprehensive behavioral analysis. Myelin ultrastructure and the expression of myelin-specific proteins are also investigated. The present study reveals slight differences with respect to behavioral effects of Ag(+)- but not nanoAg-treated rats. A weak depressive effect and hyperalgesia were observed after Ag(+) exposure whereas administration of nanoAg was found to specifically increase body weight and body temperature of animals. Both nanoAg and Ag(+) induce morphological disturbances in myelin sheaths and alter the expression of myelin-specific proteins CNP, MAG and MOG. These results suggest that the CNS may be a target of low-level toxicity of nanoAg.

µ-Opioid receptor transcriptional variants in the murine forebrain and spinal cord.

Oprm1, the gene encoding the µ-opioid receptor, has multiple reported transcripts, with a variable 3' region and many alternative sequences encoding the C-terminus of the protein. The functional implications of this variability remain mostly unexplored, though a recurring notion is that it could be exploited by developing selective ligands with improved clinical profiles. Here, we comprehensively examined Oprm1 transcriptional variants in the murine central nervous system, using long-read RNAseq as well as spatial and single-cell transcriptomics. The results were validated with RNAscope in situ hybridization. We found a mismatch between transcripts annotated in the mouse genome (GRCm38/mm10) and the RNA-seq results. Sequencing data indicated that the primary Oprm1 transcript has a 3' terminus located on chr10:6,860,027, which is âˆ¼ 9.5 kilobases downstream of the longest annotated exon 4 end. Long-read sequencing confirmed that the final Oprm1 exon included a 10.2 kilobase long 3' untranslated region, and the presence of the long variant was unambiguously confirmed using RNAscope in situ hybridization in the thalamus, striatum, cortex and spinal cord. Conversely, expression of the Oprm1 reference transcript or alternative transcripts of the Oprm1 gene was absent or close to the detection limit. Thus, the primary transcript of the Oprm1 mouse gene is a variant with a long 3' untranslated region, which is homologous to the human OPRM1 primary transcript and encodes the same conserved C-terminal amino acid sequence.