A surge of late-occurring meiotic double-strand breaks rescues synapsis abnormalities in spermatocytes of mice with hypomorphic expression of SPO11

Meiosis is the biological process that, after a cycle of DNA replication, halves the cellular chromosome complement, leading to the formation of haploid gametes. Haploidization is achieved via two successive rounds of chromosome segregation, meiosis I and II. In mammals, during prophase of meiosis I, homologous chromosomes align and synapse through a recombination-mediated mechanism initiated by the introduction of DNA double-strand breaks (DSBs) by the SPO11 protein. In male mice, if SPO11 expression and DSB number are reduced below heterozygosity levels, chromosome synapsis is delayed, chromosome tangles form at pachynema, and defective cells are eliminated by apoptosis at epithelial stage IV at a spermatogenesis-specific endpoint. Whether DSB levels produced in Spo11 +/− spermatocytes represent, or approximate, the threshold level required to guarantee successful homologous chromosome pairing is unknown. Using a mouse model that expresses Spo11 from a bacterial artificial chromosome, within a Spo11 −/− background, we demonstrate that when SPO11 expression is reduced and DSBs at zygonema are decreased (approximately 40 % below wild-type level), meiotic chromosome pairing is normal. Conversely, DMC1 foci number is increased at pachynema, suggesting that under these experimental conditions, DSBs are likely made with delayed kinetics at zygonema. In addition, we provide evidences that when zygotene-like cells receive enough DSBs before chromosome tangles develop, chromosome synapsis can be completed in most cells, preventing their apoptotic elimination

Tumor necrosis factor-alpha - mediated 2-hydroxyethyl methacrylate cytotoxic and inflammatory effect on human gingival fibroblasts

2-Hydroxyethyl methacrylate (HEMA), deriving from polymerized dental resinous biomaterials, can diffuse throughout the dentin organic matrix, preventing collagen collapse, but also at gingival and tooth pulp level [1]. HEMA could induce toxic effects, such as tissue inflammation, also at relatively low concentrations. Our study aimed to investigate the cytotoxic and inflammatory effect exerted on human gingival fibroblasts (HGFs) by a low HEMA concentration evaluating cell viability by Trypan blue dye exclusion test, early apoptosis and reactive oxygen species (ROS) production by flow cytometry and gene expression of specific proteins involved in the inflammatory process, such as tumor necrosis factor-alpha (TNF-α) and cyclooxygenase-2 (COX-2), by real-time reverse transcription polymerase chain reaction (real-time RT PCR). Cultured HGFs, obtained from fragments of gingival tissue, were exposed to 3 mM HEMA in Dulbecco’s modified Eagle’s medium for 0, 24 or 96 hours. In our experimental model, both 24- and 96-hour HEMA treatment decreased cell viability of about 20%. In parallel Annexin-V/PI assay, which detects apoptosis, indicated a 18% of Annexin-V positive cells after 24- and 96-hour HEMA incubation. After 24-hour HEMA treatment we observed an increase of ROS persisting up to 96 hours. Interestingly, 24-hour HEMA treatment increased TNF-α gene expression of about 80% and COX-2 mRNA levels of about 70% compared to control. After 96-hour HEMA incubation, TNF-α gene expression was about sixfold and COX-2 mRNA levels were about fivefold compared to control. Increase of TNF-α and COX-2 gene expression was hence HEMA exposure time-dependent. Since TNF-α - induced inflammation has been shown to be mediated by the activation of COX-2 transcription in HGFs [2], we can hypothesize that, in our experimental model, 24- or 96-hour HEMA treatment in HGFs induces a ROS-mediated cytotoxicity and an inflammatory process modulated by increase of TNF-α gene expression, which could rapidly produce the observed up-regulation of COX-2 transcription. Thus, the knowledge of molecular mechanisms underlying cellular response to dental resinous biomaterials, identifying threshold over which these compounds become toxic, could allow to set up protocols for a more effective clinical practice and for a better performance of tested materials. [1] Schweikl H, Spagnuolo G, Schmalz G. J Dent Res 2006; 85: 870-7. [2] Nakao S, Ogata Y, Shimizu E, Yamazaki M, Furuyama S, Sugiya H. Mol Cell Biochem 2002; 238: 11-8

G9a co-suppresses LINE1 elements in spermatogonia

BACKGROUND: Repression of retrotransposons is essential for genome integrity and the development of germ cells. Among retrotransposons, the establishment of CpG DNA methylation and epigenetic silencing of LINE1 (L1) elements and the intracisternal A particle (IAP) endogenous retrovirus (ERV) is dependent upon the piRNA pathway during embryonic germ cell reprogramming. Furthermore, the Piwi protein Mili, guided by piRNAs, cleaves expressed L1 transcripts to post-transcriptionally enforce L1 silencing in meiotic cells. The loss of both DNA methylation and the Mili piRNA pathway does not affect L1 silencing in the mitotic spermatogonia where histone H3 lysine 9 dimethylation (H3K9me2) is postulated to co-repress these elements. RESULTS: Here we show that the histone H3 lysine 9 dimethyltransferase G9a co-suppresses L1 elements in spermatogonia. In the absence of both a functional piRNA pathway and L1 DNA methylation, G9a is both essential and sufficient to silence L1 elements. In contrast, H3K9me2 alone is insufficient to maintain IAP silencing in spermatogonia. The loss of all three repressive mechanisms has a major impact on spermatogonial populations inclusive of spermatogonial stem cells, with the loss of all germ cells observed in a high portion of seminiferous tubules. CONCLUSIONS: Our study identifies G9a-mediated H3K9me2 as a novel and important L1 repressive mechanism in the germ line. We also demonstrate fundamental differences in the requirements for the maintenance of L1 and IAP silencing during adult spermatogenesis, where H3K9me2 is sufficient to maintain L1 but not IAP silencing. Finally, we demonstrate that repression of retrotransposon activation in spermatogonia is important for the survival of this population and testicular homeostasis

The RNA m6A Reader YTHDF2 Is Essential for the Post-transcriptional Regulation of the Maternal Transcriptome and Oocyte Competence.

YTHDF2 binds and destabilizes N6-methyladenosine (m6A)-modified mRNA. The extent to which this branch of m6A RNA-regulatory pathway functions in vivo and contributes to mammalian development remains unknown. Here we find that YTHDF2 deficiency is partially permissive in mice and results in female-specific infertility. Using conditional mutagenesis, we demonstrate that YTHDF2 is autonomously required within the germline to produce MII oocytes that are competent to sustain early zygotic development. Oocyte maturation is associated with a wave of maternal RNA degradation, and the resulting relative changes to the MII transcriptome are integral to oocyte quality. The loss of YTHDF2 results in the failure to regulate transcript dosage of a cohort of genes during oocyte maturation, with enrichment observed for the YTHDF2-binding consensus and evidence of m6A in these upregulated genes. In summary, the m6A-reader YTHDF2 is an intrinsic determinant of mammalian oocyte competence and early zygotic development

Effects of methacrilyc thermosets coated with Silver-polysaccharide nanocomposite on HGFs adhesion in a S. mitis co-culture system

Silver based medical products have been proven to be effective in retarding and preventing bacterial growth, being silver reported to control infections since ancient times (1). In the field of dentistry, the use of silver ions/nanoparticles has been explored to counteract bacteria in resins and implants, as silver can destroy bacterial cell walls by reacting with the thiol groups (–SH) of proteins exposed to the extracellular portion of the bacterial membrane. Conversely, eukaryotic cells lack these exterior binding sites, so nanoparticles are supposed to interact with them only upon metal internalization (2). To reduce both bacterial adhesion to dental devices and cytotoxicity against eukaryotic cells, we coated BisGMA/TEGDMA methacrylic thermosets with a new material, Chitlac-nAg, formed by stabilized silver nanoparticles with a polyelectrolyte solution containing Chitlac. Here we analyzed the proliferative and adhesive ability of human gingival fibroblasts (HGFs) on BisGMA/TEGDMA thermosets uncoated and coated with AgNPs in a co-culture model system with Streptococcus mitis. After 48 h, HGFs well adhered onto both surfaces, while S. mitis cytotoxic response was higher in the presence of AgNPs coated thermosets. After 24 h thermosets coated with Chitlac as well as those coated with Chitlac-nAg exerted a minimal cytotoxic effect on HGFs, while after 48 h LDH release rised up to 20%. Moreover, the presence of S. mitis reduced this release mainly when HGFs adhered to Chitlac-nAg coated thermosets. The reduced secretion of collagen type I was significant in the presence of both surfaces even more when saliva is added. Integrin β1 localized closely to cell membranes onto Chitlac-nAg thermosets and PKC α translocated into nuclei. These data confirm that Chitlac-nAg thermosets have a promising utilization in the field of restorative dentistry exerting their antimicrobial activity due to AgNPs without cytotoxicity for eukaryotic cells.This work was supported by grants from MIUR FIRB 2010 and MIUR PRIN-2009

Reduction of mdx mouse muscle degeneration by low-intensity endurance exercise: a proteomic analysis in quadriceps muscle of exercised versus sedentary mdx mice

In our recent study was shown a significant recovery of damaged skeletal muscle of mice with x-linked muscular dystrophy (mdx) following low-intensity endurance exercise, probably by reducing the degeneration of dystrophic muscle. Consequently, in the present work we aimed to identify proteins involved in the observed reduction of degenerating fibers. To this end, we used proteomic analysis to evaluate changes in the protein profile of quadriceps dystrophic muscles of exercised versus sedentary mdx mice. Four protein spots were found to be significantly changed and were identified as three isoforms of Carbonic anhydrase 3 (CA3) and superoxide dismutase [Cu-Zn] (SODC). Protein levels of CA3 isoforms were significantly up-regulated in quadriceps of sedentary mdx mice and were completely restored to wild type mice values, both sedentary and exercised, in quadriceps of exercised mdx mice. Protein levels of SODC were down-regulated in quadriceps of sedentary mdx mice and were significantly restored to wild type mice values, both sedentary and exercised, in quadriceps of exercised mdx mice. Western blot data were in agreement with those obtained using proteomic analysis and revealed the presence of one more CA3 isoform that was significantly changed. Based on data found in the present study, it seems that low-intensity endurance exercise may in part contribute to reduce cell degeneration process in mdx muscles, by counteracting oxidative stress

Escape from cell death through authophagy in Human Gingival Fibroblast/Streptococcus mitis co-culture treated with Chitlac n-Ag

Since ancient times, silver has been extensively used to control infections. Silver based medical products have been proved to be effective in retarding and preventing bacterial infections (Chen et al., 2007). In order to prevent silver nanoparticles aggregation a lactose-modified chitosan was shown to be effective in stabilizing colloidal solutions of silver nanoparticles: “Chitlac-nAg” (Travan et al., 2009). Silver ions and nanoparticle are capable to destroy the bacterial cell wall by reacting with sulfhydryl groups on membrane proteins (Kruszewski et al., 2003). Since the cells are capable of internalizing nanoparticles there is the risk of a massive uptake by eukaryotic cells, which eventually leads to their death through oxidative DNA damage (Li et al. 2013) In the present work we investigated the effects of Chitlac-nAg on primary human gingival fibroblast (HGFs) co-cultured with Streptococcus mitis in the presence of saliva. HGFs were obtained from fragments of healty marginal gingival tissue, co-cultured with the clinical strain of S. mitis and treated for 24-48h with Chitlac or Chitlac-nAg. Cytotoxicity evaluated by LDH assay showed an increment in LDH release in co-culture in the presence of Chitlac n-Ag and saliva. Oxidative stress detected by means of Reactive Oxygen Species formation highlighted an early ROS presence in samples with Chitlac-nAg and saliva, but this value was similar to control after 48h; apoptotic and necrotic cells were detected by means of Annexin V/PI showing an increase in cell death in HGFs treated with Ag and saliva after 24h, and returned to basal levels after 48h; the uptake of nanoparticles by cells was determined by optical and electronic microscopy revealing the Ag uptake in vesicles. The presence of lysosomes and autophagosomes was verified by Lysotracker and by LC3 respectively. In vitro results showed that in our co-culture model, which mimics the microenvironment of the oral cavity, chitlac n-Ag does not exert cytotoxic effect towards HGFs that are able to execute a homeostasis mechanism through autophagy promoting cell survival