138 Combination of protein synthesis and phosphorylation inhibitors on the activation of bovine oocytes

Oocyte activation combining protein synthesis (cycloheximide [CHX]) and phosphorylation inhibitors (6-dimethylaminopurine [DMAP]) has improved the in vitro embryo production rates by intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT), respectively, in different species. Thus, our aim was to study the effect of combinations of inhibitors of protein synthesis and phosphorylation to activate bovine oocytes, including in these combinations anisomycin (ANY), a protein synthesis inhibitor, the positive effect of which in oocyte activation we previously reported, which was also used as the control (ANY). The combined activation treatments included ANY + CHX, ANY + DMAP, ANY + CHX + DMAP, and CHX + DMAP. We assessed the cleavage and blastocyst rate by stereomicroscope observation at 72 and 168 h, respectively. Additionally, we evaluated by Western blotting analysis, the effects on the maturation-promoting factor (MPF) components, including the level of CDK1 and phosphorylation level of its inhibitory and activating residues, and the ERK1/2 MAPK pathways by the level of ERK1/2 and its phosphorylation profile. The differential expression of embryo quality genes by RT-qPCR was also assessed. The proportional data of the embryonic development were subjected to an arcsine transformation. Both the transformed values and the values normalised of the Western blotting and the abundance of genes were analysed by one-way analysis of variance (ANOVA). The analysis of variation on the phosphorylation levels of CDK1 residues was done using a two-way ANOVA. The post hoc analysis was carried out using the Scheffe test. P-values ≤ 0.05 were considered statistically significant. The results showed that cleavage and blastocyst rates (mean: 95.2% and 46.3%, respectively), as well as the expression of CYCLIN B1, CDK1, p-CDK1^Thr161, and p-CDK1^Thr14-Tyr15 were similar among groups. ANY and ANY + CHX reduced the expression of ERK1/2 compared with DMAP, whereas ANY + DMAP, CHX + DMAP, and ANY + CHX + DMAP reduced p-ERK1/2 compared with ANY and ANY + CHX. ANY and ANY + CHX reduced the expression of ERK1/2 compared with treatments with DMAP, whereas ANY + DMAP, CHX + DMAP and ANY + CHX + DMAP reduced p-ERK1/2 compared with ANY and ANY + CHX. Gene expression analyses in blastocysts showed higher levels of OCT4 in embryos activated with ANY + CHX + DMAP treatment compared with other groups, while CDX2 did not show differences. The ratio of BAX and BCL2A1 was greater in the ANY + CHX + DMAP group compared with CHX + DMAP treatment; however, BAX/BCL2 ratio of ANY + CHX + DMAP treatment did not differ with ANY, ANY + CHX or ANY + DMAP. At the same line, CHX + DMAP treatment was similar to ANY, ANY + CHX and ANY + DMAP groups. In conclusion, oocyte activation by the dual inhibition of protein synthesis and phosphorylation induces MPF inactivation without degradation of CYCLIN B1, while MAPK inactivation occurs differentially between these inhibitors. Thus, although the combined use of these inhibitors does not affect early embryo development, it positively impacts the expression of genes associated with embryo quality.

Funding support from ANID/CONICYT Chile grant FONDECYT 1181453 is gratefully acknowledged.