Effects of epigallocatechin-3-gallate on bovine oocytes matured

Objective Epigallocatechin-3-gallate (EGCG) is a major ingredient of catechin polyphenols and is considered one of the most promising bioactive compounds in green tea because of its strong antioxidant properties. However, the protective role of EGCG in bovine oocyte in vitro maturation (IVM) has not been investigated. Therefore, we aimed to study the effects of EGCG on IVM of bovine oocytes. Methods Bovine oocytes were treated with different concentrations of EGCG (0, 25, 50, 100, and 200 μM), and the nuclear and cytoplasmic maturation, cumulus cell expansion, intracellular reactive oxygen species (ROS) levels, total antioxidant capacity, the early apoptosis and the developmental competence of in vitro fertilized embryos were measured. The mRNA abundances of antioxidant genes (nuclear factor erythriod-2 related factor 2 [NRF2], superoxide dismutase 1 [SOD1], catalase [CAT], and glutathione peroxidase 4 [GPX4]) in matured bovine oocytes were also quantified. Results Nuclear maturation which is characterized by first polar body extrusion, and cytoplasmic maturation characterized by peripheral and cortical distribution of cortical granules and homogeneous mitochondrial distribution were significantly improved in the 50 μM EGCG-treated group compared with the control group. Adding 50 μM EGCG to the maturation medium significantly increased the cumulus cell expansion index and upregulated the mRNA levels of cumulus cell expansion-related genes (hyaluronan synthase 2, tumor necrosis factor alpha induced protein 6, pentraxin 3, and prostaglandin 2). Both the intracellular ROS level and the early apoptotic rate of matured oocytes were significantly decreased in the 50 μM EGCG group, and the total antioxidant ability was markedly enhanced. Additionally, both the cleavage and blastocyst rates were significantly higher in the 50 μM EGCG-treated oocytes after in vitro fertilization than in the control oocytes. The mRNA abundance of NRF2, SOD1, CAT, and GPX4 were significantly increased in the 50 μM EGCG-treated oocytes. Conclusion In conclusion, 50 μM EGCG can improve the bovine oocyte maturation, and the protective role of EGCG may be correlated with its antioxidative property

Sulforaphane suppresses paraquat-induced oxidative damage in bovine in vitro-matured oocytes through Nrf2 transduction pathway

Sulforaphane (SFN), a bioactive phytocompound extracted from cruciferous plants, has received increasing attention due to its vital cytoprotective role in eliminating oxidative free radical through activation of nuclear factor erythroid 2-related factor (Nrf2)-mediated signal transduction pathway. This study aims at a better insight into the protective benefit of SFN in attenuating paraquat (PQ)-caused impairment in bovine in vitro-matured oocytes and the possible mechanisms involved therein. Results showed that addition of 1 μM SFN during oocyte maturation obtained higher proportions of matured oocytes and in vitro-fertilized embryos. SFN application attenuated the toxicological effects of PQ on bovine oocytes, as manifested by enhanced extending capability of cumulus cell and increased extrusion proportion of first polar body. Following incubation with SFN, oocytes exposed to PQ exhibited reduced intracellular ROS and lipid accumulation levels, and elevated T-SOD and GSH contents. SFN also effectively inhibited PQ-mediated increase in BAX and CASPASE-3 protein expressions. Besides, SFN promoted the transcription of NRF2 and its downstream antioxidative-related genes GCLC, GCLM, HO-1, NQO-1, and TXN1 in a PQ-exposed environment, indicating that SFN prevents PQ-caused cytotoxicity through activation of Nrf2 signal transduction pathway. The mechanisms underlying the role of SFN against PQ-induced injury included the inhibition of TXNIP protein and restoration of the global O-GlcNAc level. Collectively, these findings provide novel evidence for the protective role of SFN in alleviating PQ-caused injury, and suggest that SFN application may be an efficacious intervention strategy against PQ cytotoxicity

Knockdown of CLAUDIN-6 Inhibited Apoptosis and Induced Proliferation of Bovine Cumulus Cells

This study aims to investigate the effects of CLAUDIN-6 (CLDN6) on cell apoptosis and proliferation of bovine cumulus cells (CCs). Immunofluorescence staining was used to localize CLDN6 protein in CCs. Three pairs of siRNA targeting CLDN6 and one pair of siRNA universal negative sequence as control were transfected into bovine CCs. Then, the effective siRNA was screened by real-time quantitative PCR (RT-qPCR) and Western blotting. The mRNA expression levels of apoptosis related genes (CASPASE-3, BAX and BCL-2) and proliferation related genes (PCNA, CDC42 and CCND2) were evaluated by RT-qPCR in CCs with CLDN6 knockdown. Cell proliferation, apoptosis and cell cycle were detected by flow cytometry with CCK-8 staining, Annexin V-FITC staining and propidium iodide staining, respectively. Results showed that the CLDN6 gene was expressed in bovine CCs and the protein was localized in cell membranes and cytoplasms. After CLDN6 was knocked down in CCs, the cell apoptosis rate significantly decreased and the pro-apoptotic genes BAX and CASPASE-3 were down-regulated significantly, whereas the anti-apoptotic gene BCL-2 was markedly up-regulated (p PCNA, CCND2 and CDC42 increased obviously in CCs with CLDN6 knockdown (p p p > 0.05). Therefore, the expression of CLDN6 and its effects on cell proliferation, apoptosis and cell cycle of bovine CCs were first studied. CLDN6 low expression inhibited cell apoptosis, induced cell proliferation and cell cycle arrest of bovine CCs

Dosimetric analysis of rib interference of the CTV during interstitial brachytherapy of lung tumors

Purpose : In interstitial brachytherapy for lung tumors, the placement and alignment of the source needles are influenced by the ribs, which can affect the dose distribution. This study evaluated the change in dose to the target by comparing the dose between the actual interstitial brachytherapy plan (AIBP, what is deliverable due to anatomic constraints), and the virtual interstitial brachytherapy plan (VIBP, pretreatment-modified dose distribution). Material and methods : AIBPs and VIBPs were designed for 20 lung tumors. The VIBP was designed with uniform spacing between needles, regardless of the presence of ribs. The prescription dose was 30 Gy. The percentage of normal ipsilateral lung volume that received a dose ≥ 5 Gy (V 5 ), conformity index (COIN), incremental dose percentage (IDP) to the target, and the dose covering 95% (D 95 ) of the clinical target volume (CTV) were calculated. Results : The V 5 of the VIBPs was significantly smaller than that of the AIBPs (p < 0.01). The mean COIN value was 0.41 ± 0.12 for the AIBPs, which was significantly smaller than the value 0.54 ± 0.12 for the VIBPs (p < 0.01). The D 95 of CTV in VIBP-adjusted was greater than that in AIBPs (p < 0.01). The mean IDP was 44% ± 40%. The D max of the ribs was 20.16 Gy ± 15.76 Gy in AIBPs, and 18.57 Gy ± 15.14 Gy in VIBPs, which was not significantly different (p > 0.05). Conclusions : The regular geometric alignment of needles is important for increasing the target dose and limiting the normal lung dose in interstitial brachytherapy for thoracic tumors. Thus, we recommend that radiation oncologists attempt to achieve the regular alignment of needles during implantation

Identification of the porcine IG-DMR and abnormal imprinting of DLK1-DIO3 in cloned pigs.

Correct reprogramming of the DLK1-DIO3 imprinted region is critical for the development of cloned animals. However, in pigs, the imprinting and regulation of the DLK1-DIO3 region has not been systematically analyzed. The objective of this study was to investigate the imprinting status and methylation regulation of the DLK1-DIO3 region in wild-type and cloned neonatal pigs. We mapped the imprinting control region, IG-DMR, by homologous alignment and validated it in sperm, oocytes, fibroblasts, and parthenogenetic embryos. Subsequently, single nucleotide polymorphism-based sequencing and bisulfite sequencing polymerase chain reaction were conducted to analyze imprinting and methylation in different types of fibroblasts, as well as wild-type and cloned neonatal pigs. The results showed that Somatic cell nuclear transfer (SCNT) resulted in hypermethylation of the IG-DMR and aberrant gene expression in the DLK1-DIO3 region. Similar to wild-type pigs, imprinted expression and methylation were observed in the surviving cloned pigs, whereas in dead cloned pigs, the IG-DMR was hypermethylated and the expression of GTL2 was nearly undetectable. Our study reveals that abnormal imprinting of the DLK1-DIO3 region occurs in cloned pigs, which provides a theoretical basis for improving the cloning efficiency by gene editing to correct abnormal imprinting

Efficient Editing of the ZBED6-Binding Site in Intron 3 of IGF2 in a Bovine Model Using the CRISPR/Cas9 System

Background: Insulin-like growth factor 2 is a growth-promoting factor that plays an important role in the growth and development of mammals. A nucleotide substitution in intron 3 of IGF2&mdash;which disrupts the ZBED6-binding site&mdash;affects muscle mass, organ size, and fat deposition in pigs. The ZBED6-binding site is also conserved in cattle. Methods: In the present study, we introduced mutations in the ZBED6-binding site in intron3 of IGF2 in bovine fetal fibroblasts using the CRISPR/Cas9 system, and investigated the effect of disruption of ZBED6 binding on IGF2 expression. Results: Eleven biallelic-mutant single-cell clones were established, three of which contained no foreign DNA residues. Single-cell clones 93 and 135 were used to produce cloned embryos. Dual-luciferase reporter assay in C2C12 cells demonstrated that the mutation in the ZBED6-binding site increases the promoter 3 activity of bovine IGF2. A total of 49 mutant cloned embryos were transplanted into surrogate cows. Unfortunately, all cloned embryos died before birth. IGF2 was found to be hypomethylated in the only fetus born (stillborn), which may have been due to the incomplete reprogramming. Conclusions: We efficiently constructed IGF2-edited cell lines and cloned embryos, which provided a theoretical basis and experimental materials for beef cattle breeding

Silicone rubber aging in electrolyzed aqueous salt environments

In this work we suggest an entirely new and highly transformative model of silicone rubber aging caused by the presence of electrolyzed aqueous salt, leading to the formation of hypochlorous acid in-service on energized High Voltage (HV) transmission lines in coastal environments. The model was subsequently verified by aging one-component Room Temperature Vulcanized (RTV-1) rubbers in commercially produced hypochlorous acid and an electrolyzed aqueous salt environment at room temperature. Molecular dynamics models of the rubbers with silica fillers were conducted to offer at least partial explanations of the types and extents of aging observed in the RTV-1 samples tested in the aqueous salt solutions. We have shown that the above environments are highly damaging to the silicone rubber polymer network and significantly more destructive than non-electrolyzed standard aqueous salt solutions at higher temperatures. The main observations of this research should greatly contribute to a better understanding of complex degradation mechanisms of modern HV silicone rubber compounds, which are utilized in some of the most critical lines and substations all over the world