Alteration of gene expression profile in mouse embryonic stem cells and neural differentiation deficits by ethephon.

Ethephon, a member of the organophosphorus compounds, is one of the most widely used plant growth regulators for artificial ripening. Although million pounds of this chemical is being used annually, the knowledge regarding its molecular toxicity is yet not sufficient. The purpose of this study was to evaluate the potential developmental toxicity of ethephon using embryonic stem cell model. The mouse embryonic stem cells (mESCs) were exposed to various concentrations of ethephon and the viability, cell cycle alteration and changes in the gene expression profile were evaluated using high-throughput RNA sequencing. Further, the effect of ethephon on neural differentiation potential was examined. The results showed that ethephon at noncytotoxic doses induced cell cycle arrest in mESCs. Gene ontology enrichment analysis showed that terms related to cell fate and organismal development, including neuron fate commitment, embryo development and cardiac cell differentiation, were markedly enriched in ethephon-treated cells. Neural induction of mESCs in the presence of ethephon was inhibited and the expression of neural genes was decreased in differentiated cells. Results obtained from this work clearly demonstrate that ethephon affects the gene expression profile of undifferentiated mESCs and prevents neural differentiation. Therefore, more caution against the frequent application of ethephon is advised.

Urokinase-plasminogen activator protects periodontal ligament fibroblast from oxidative induced-apoptosis and DNA damage.

BACKGROUND AND OBJECTIVE: Urokinase-plasminogen activator (uPA) is a serine protease expressed at high basal level in normal gingival cervical fluid. Despite its known pathologic role in tissue proteolysis in periodontitis, little is known concerning uPA physiological function in oral tissue. Recent evidence in cancer cells has implicated the uPA system in DNA repair and anti-apoptotic pathways. This study is aimed to evaluate the protective function of urokinase against oxidative DNA damage in periodontal ligament (PDL) fibroblast, and to propose a new biological role for uPA in oral cavity. MATERIAL AND METHODS: PDL cells were isolated from human wisdom teeth obtained from healthy donors. An oxidative stress model was created in which PDL cells were incubated with 20, 30, 40 and 60 µmol/L hydrogen peroxide. Twenty-four hours before and after peroxide treatment, cells were treated with uPA and amiloride. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide assay, apoptosis by DAPI-staining and annexin V/propidium iodide assay, and DNA breaks by alkaline comet assay. For estimating DNA damage level, γ-H2AX expression was studied using flow cytometry and immunostaining. RESULTS: The incubation of the peroxide-treated cells with uPA significantly increased cell viability and decreased apoptosis. A significant decrease in the number of γ-H2AX foci was seen at 30 µmol/L hydrogen peroxide in uPA-treated cells. uPA inhibition as a result of amiloride treatment, in turn, induced a reduction in cell viability. In addition, there was a significant decrease in the levels of DNA damage in uPA-treated groups as measured by the comet assay. CONCLUSION: The present study brings support to the theory that uPA may have a protective role for periodontal tissue and could protect PDL fibroblasts from oxidative DNA damage and apoptosis.

Increased sperm ubiquitination correlates with abnormal chromatin integrity.

Ubiquitin, a 8.5 kDa peptide that marks other proteins for proteasomal degradation, tags defective spermatozoa during epididymal passage and is proposed as a biomarker for sperm quality. The present study was designed to evaluate the relationships between sperm ubiquitination, sperm chromatin integrity and semen parameters. Semen samples from 63 couples were collected and analysed according to World Health Organization criteria. Each sample was evaluated for sperm ubiquitination by the direct immunofluorescence method, using anti-ubiquitin antibodies. Chromatin integrity of the same samples was analysed using acridine orange (AO) and toluidine blue (TB) tests. A positive correlation was found between ubiquitinated spermatozoa and the percentage of spermatozoa with abnormal chromatin (AO: r = 0.58, P < 0.001 and TB: r = 0.48, P < 0.001). Negative correlations were obtained between sperm ubiquitination and: sperm count (r = -0.2, P = 0.048), sperm morphology (r = -0.36, P = 0.003), rapidly progressive motility (r = -0.25, P = 0.044) and slow progressive motility (r = -0.28, P = 0.022). Sperm ubiquitination was positively correlated with the percentage of immotile spermatozoa. These results show that among semen parameters, chromatin abnormality is more closely associated with sperm ubiquitination and further validate sperm ubiquitination as a suitable marker for sperm quality.