81 IMPROVEMENT OF DEVELOPMENTAL COMPETENCE OF BOVINE IN VITRO-PRODUCED EMBRYOS BY ADDING 2-METHOXYSTYPANDRONE IN MATURATION MEDIA

The 2-methoxystypandrone (2-MS) is a naphthoquinone isolated from Polygonum cuspidatum. The objective of this study was to investigate the effects of 2-MS on oocyte maturation, blastocyst development, and embryo quality in terms of cell number and gene expression in vitro. A total of 2364 oocytes were cultured in TCM-199 supplemented with 10% fetal bovine serum, 1 μg mL-1 oestradiol-17β, 10 μg mL-1 FSH, 10 ng mL-1 epidermal growth factor, 0.6 mM cysteine, and 0.2 mM sodium pyruvate and supplemented with different concentrations of 2-MS as following: 1.5 μM (n = 458), 1.0 (n = 493), 0.5 (n = 468), 0.1 (n = 470), and 0 μM (control, n = 475) followed by IVF and then culture in CR1-aa medium supplemented with 44 μg mL-1 sodium pyruvate, 14.6 μg mL-1 glutamine, 10 μL mL-1 penicillin-streptomycin, 3 mg mL-1 BSA, and 310 μg mL-1 glutathione for the first 3 days, and then the BSA was replaced with 10% FBS until Day 8. The differences in embryo development between experimental groups were analysed by one-way ANOVA. The Duncan’s multiple range tests were used to test the differences between the treatments. The level of statistical significance was set at P < 0.05. The results showed that the addition of 2-MS at 1.0 mM significantly improved (P < 0.05) the percentage of MII oocytes, which were identified by aceto-orcein staining, compared with that in the control (76.5 v. 65.4%, respectively), and remarkably (P < 0.05), improved blastocyst development rates (45.29%) compared with control (32.21%). Additionally, TUNEL assay demonstrated that treatment with 1.0 μM of 2-MS significantly improved the embryo quality by increasing total number of cells and reducing DNA damage. Immunofluorescent analysis showed that the protein levels of nuclear factor-kappa B (NFkB), inhibitor of kappa B kinase β (IkKβ), 8-oxoguanine, and cyclooxygenase-2 (COX2) declined significantly (P < 0.05) after 2-MS treatment compared with the control. These results were confirmed by qRT-PCR, which showed a significant decrease in the mRNA levels of NFkB, IkKβ, COX2, inducible nitric oxide synthase (iNOS), BCL2-associated X protein (BAX), caspase-3, and Janus kinase2 (JAK2) after 2-MS treatment; however, the mRNA level of the anti-apoptotic gene B-cell lymphoma2 (BCL2) was significantly higher than that in the control. In conclusion, the addition of 2-MS at the indicated concentration dramatically improves the developmental competence of bovine in vitro-produced embryos. This work was supported by a grant from the Next-Generation BiogGeen21 (No. PJ01107703), IPET (No. 315017–5), BK21plus, and KGSP.