Protective effect of vitamin C and lycopene on the in vitro fertilization capacity of sex-sorted bull sperm by inhibiting the oxidative stress.

The fertilization capacity of sex-sorted sperms is seriously decreased, which inhibits its wide application. However, little information is still available about the effect of vitamin C (VC) and lycopene (Lyc) on the fertilization capacity of sex-sorted bull sperm. In this study, the washing medium and fertilization medium of sex-sorted sperm from three bull individuals were supplemented with different concentrations of VC (0, 1 × 10-3 , 1 × 10-4 , 1 × 10-5 , 1 × 10-6  M) or Lyc (0, 1 × 10-4 , 1 × 10-5 , 1 × 10-6 , 1 × 10-7 ). After washing twice and incubation for 1.5 hr, the malondialdehyde (MDA) level, phosphatidylserine (PS) translocation, membrane potential (Δψm) and IVF (in vitro fertilization) ability of sex-sorted sperm were investigated. For the sex-sorted sperm of bulls A, B and C, 1 × 10-3  M VC or 1 × 10-4  M Lyc treatment significantly decreased their MDA levels and PS translocation and increased their Δψm levels and cleavage rates after IVF. When blastocysts were concerned, 1 × 10-4  M Lyc significantly improved the blastocyst rates and their IFN-tau expression of bulls A and C. In conclusion, supplementation of 1 × 10-3  M VC or 1 × 10-4  M Lyc in washing and fertilization medium contributed greatly to improving the fertilization capacity of sex-sorted bull sperm during IVF procedure.

Oocyte IVM or vitrification significantly impairs DNA methylation patterns in blastocysts as analysed by single-cell whole-genome methylation sequencing.

To explore the mechanisms leading to the poor quality of IVF blastocysts, the single-cell whole-genome methylation sequencing technique was used in this study to analyse the methylation patterns of bovine blastocysts derived from invivo, fresh (IVF) or vitrified (V_IVF) oocytes. Genome methylation levels of blastocysts in the IVF and V_IVF groups were significantly lower than those of the invivo group (P<0.05). In all, 1149 differentially methylated regions (DMRs) were identified between the IVF and invivo groups, 1578 DMRs were identified between the V_IVF and invivo groups and 151 DMRs were identified between the V_IVF and IVF groups. For imprinted genes, methylation levels of insulin-like growth factor 2 receptor (IGF2R) and protein phosphatase 1 regulatory subunit 9A (PPP1R9A) were lower in the IVF and V_IVF groups than in the invivo group, and the methylation level of paternally expressed 3 (PEG3) was lower in the V_IVF group than in the IVF and invivo groups. Genes with DMRs between the IVF and invivo and the V_IVF and IVF groups were primarily enriched in oocyte maturation pathways, whereas DMRs between the V_IVF and invivo groups were enriched in fertilisation and vitrification-vulnerable pathways. The results of this study indicate that differences in the methylation of critical DMRs may contribute to the differences in quality between invitro- and invivo-derived embryos.

Melatonin Improves the Fertilization Capacity of Sex-Sorted Bull Sperm by Inhibiting Apoptosis and Increasing Fertilization Capacitation via MT1.

Little information is available regarding the effect of melatonin on the quality and fertilization capability of sex-sorted bull sperm, and even less about the associated mechanism. Sex-sorted sperm from three individual bulls were washed twice in wash medium and incubated in a fertilization medium for 1.5 h, and each was supplemented with melatonin (0, 10-3 M, 10-5 M, 10-7 M, and 10-9 M). The reactive oxygen species (ROS) and endogenous antioxidant activity (glutathione peroxidase (GPx); superoxide dismutase (SOD); catalase (CAT)), apoptosis (phosphatidylserine [PS] externalization; mitochondrial membrane potential (Δψm)), acrosomal integrity events (malondialdehyde (MDA) level; acrosomal integrity), capacitation (calcium ion [Ca2+]i level; cyclic adenosine monophosphate (cAMP); capacitation level), and fertilization ability of the sperm were assessed. Melatonin receptor 1 (MT1) and 2 (MT2) expression were examined to investigate the involvement of melatonin receptors on sex-sorted bull sperm capacitation. Our results show that treatment with 10-5 M melatonin significantly decreased the ROS level and increased the GPx, SOD, and CAT activities of sex-sorted bull sperm, which inhibited PS externalization and MDA levels, and improved Δψm, acrosomal integrity, and fertilization ability. Further experiments showed that melatonin regulates sperm capacitation via MT1. These findings contribute to improving the fertilization capacity of sex-sorted bull sperm and exploring the associated mechanism.

Research of methods to detect genomic mutations induced by CRISPR/Cas systems.

The indel-forming non-homologous end joining (NHEJ) pathway repairs double strand breaks in mammalian genomes, resulting in mutation formation following genome editing. Common techniques employed to identify these mutations include the amplified fragment length polymorphism (AFLP) and SURVEYOR assays, which are time consuming, laborious, and only offer a low level of sensitivity. An alternative to these approaches, which is examined in this study, is based on the quantitative PCR high-resolution melting (qPCR-HRM) curve analysis technique and offers simple implementation, is capable of handling large sample sizes, takes no more than 90 min, and produces sensitive results. Using the newly discovered RNA-guided CRISPR/Cas systems, the IL2RG and EMX1 genes were edited in the human 293T cell line in order to compare the mutation detection accuracies of the aforementioned methods. Genomic mutations were simulated by mixing mutated DNA fragments with normal fragments along a concentration gradient. The results of this comparative study showed that the HRM approach was both reproducible and accurate.

Effect of trichostatin a on SGC-7901 gastric cancer cells.

AIM: To explore Trichostatin A (TSA) effect on SGC-7901 gastric cancer cells. METHODS: MTT, fluorescence microscopy, and flow cytometry were used to assess TSA effect on cell growth and apoptosis in SGC-7901. Immunocytochemistry was used to evaluate the expression of acetylated histone H4 in SGC-7901 cells.Gene expression profile was determined by microarray assays. Glycoprotein hormones alpha subunit (CGA) gene and protein expressions in SGC-7901 cells were evaluated by Real-time PCR and Western blot, respectively. In addition, CGA protein levels in gastric adenocarcinoma and normal adjacent tissues were assessed by immunohistochemistry. RESULTS: TSA inhibited SGC-7901 cell growth. In addition, cell proliferation was significantly decreased (P = 0.02) in TSA treatment groups (0.93 ± 0.07) compared with controls (1.15 ± 0.07). Apoptosis related morphological changes, including nuclear chromatin condensation and fluorescence strength, were observed by fluorescence microscopy. These findings corroborated the increased expression of acetylated histone H4 observed in TSA treated cells compared to controls, as determined by immunocytochemistry. Interestingly, treatment of SGC-7901 cells with TSA (75 ng/ml) resulted in CGA gene down-regulation (P = 0.0381). Accordingly, CGA protein levels were decreased in TSA treated SGC-7901 cells. Finally, immunohistochemistry analysis showed that CGA expression was significantly higher in gastric adenocarcinoma tissues than normal adjacent tissues (P = 0.001). CONCLUSION: TSA induces cell apoptosis and increases the levels of acetylated histone H4 in SGC-7901 cells. In addition, TSA treatment decreases the expression in gastric cancer cells of the CGA gene, which is upregulated in gastric adenocarcinoma tissues.

Differential expression of glycoprotein non-metastatic melanoma protein B (GPNMB) involved in trichostatin A-induced apoptosis in gastric cancer.

In this study, we investigated the effect of trichostatin A (TSA) on the gastric cancer cell line BGC-823. The effect of TSA on growth inhibition and apoptosis of BGC-823 cells was examined. The gene expression profile was determined by microarray. Western blotting was used to study the levels of acetylated histone H4 and Glycoprotein non-metastatic melanoma protein B (GPNMB) proteins. GPNMB gene expression was measured by real-time PCR. GPNMB protein levels in gastric adenocarcinoma tissues and adjoining normal tissues were detected by immunohistochemistry. The results showed that a significant decrease in cell population following treatment with 75 ng/mL TSA for 48 h (0.87 ± 0.04) as compared to control (1.14 ± 0.06) (P = 0.02). Apoptotic cells were increased in TSA (75 ng/mL for 48 h) treated group as compared to the control group (from 2.02% to 19.74%) by flow cytometry. The expression of acetylated histone H4 was increased in TSA treated (75 ng/mL for 48 h) group (from 1.00 ± 0.26 to 1.87 ± 0.33, F = 5.862, P = 0.0038) as compared to the control group by Western blotting. After 48 h TSA treatment (75 ng/mL), BGC-823 cells showed decrease in GPNMB gene expression (from 1.00 ± 0.21 to 0.59 ± 0.11, F = 6.214, P = 0.0018). Immunohistochemistry showed that GPNMB expression in gastric adenocarcinoma was significantly higher than the adjoining normal tissues (P = 0.000). To conclusion, our results support that TSA can induce apoptosis, and increase acetylated histone H4 in BGC-823 cells. GPNMB expression is decreased in BGC-823 cells after TSA treatment. GPNMB is overexpressed in gastric adenocarcinoma tissue. GPNMB involved in TSA-induced apoptosis might participate in gastric cancer.