102 Expression pattern of cytoplasmic polyadenylation element binding protein 1 in bovine oocytes and embryos

Embryo development failure has often been attributed to defects in oocyte cytoplasmic maturation. IVM has been shown to cause nuclear and cytoplasmic maturation to occur asynchronously, which can lead to improper embryo development. Cytoplasmic polyadenylation element binding protein 1 (CPEB1) mediates cytoplasmic maturation, which is essential for oocyte development. However, the expression pattern of CPEB1 during bovine early embryo development and the correlation between oocyte quality and CPEB1 expression in oocytes and embryos are still unknown. The aims of this study were to determine (1) CPEB1 expression in bovine oocytes of different quality and at different stages of embryonic development following IVF, and (2) the effect of CPEB1 supplementation on bovine embryo development. Brilliant cresyl blue (BCB) staining was used as a selection method for sorting high (BCB+) and low (BCB-) quality oocytes. Cumulus–oocyte complexes (COCs) were stained with 26 mM BCB dye for 90 min at 38.5°C and sorted based on the cytoplasm coloration before IVM (Keim et al. 2023 Reprod. Fertil Dev. 35, 575–588). After 21–22 h of maturation, IVF was performed following our standard IVF protocol (Keim et al. 2023), and embryos from BCB+ and BCB- groups were cultured for 8 days in SOF medium under mineral oil. In Exp. 1, the CPEB1 expression level was evaluated via immunofluorescence staining using anti-CPEB1 antibody and AF488 secondary antibody at the MII oocyte and the 2-, 8-, and 16-cell stage and blastocyst stage embryos from both BCB+ and BCB- groups (three replicates each). In Exp. 2, CPEB1 (1579 nt, 100 ng mL⁻¹) and GFP (50 ng mL⁻¹) in vitro transcript mRNA were injected into IVF embryos at the single-cell stage and cultured for 8 days, while embryos injected with only GFP mRNA were used as controls. Cleavage and blastocyst rates were assessed at Days 2 and 8, respectively. Fluorescent intensity of CPEB1 in the oocytes and embryos was analyzed by ImageJ. Data were analyzed using generalized linear model. Differences were deemed significant if P < 0.05. Our results showed that the blastocyst rate in BCB+ group was higher than in BCB- group (20.3% vs. 10.2%, 15 replicates; P = 0.001), but there was no difference in the cleavage rate. CPEB1 levels were significantly lower in the 2-, 8-, and 16-cell stages of embryos compared with MII oocytes in both the BCB+ and BCB- groups (P < 0.001). The CPEB1 level was not detectable in blastocysts derived from either the BCB+ or BCB- group. With regard to the CPEB1 levels at each stage, a higher CPEB1 level in the BCB+ group was only observed at the 2-cell stage compared with the BCB- group (P = 0.022), but not in MII oocytes or other stages of embryos. Preliminary results of CPEB1 supplementation showed a tendency for increased blastocyst rates in CPEB1-injected embryos (n = 26) compared with control embryos (n = 26) in both the BCB+ (55.6% vs. 33.3%) and BCB- (30% vs. 20%) groups. In conclusion, a general decrease in CPEB1 expression levels occur from MII in oocytes to blastocyst stage, and oocytes of higher quality are associated with higher CPEB1 levels at the 2-cell stage in IVF embryos. Our preliminary results indicate that CPEB1 mRNA injection could improve blastocyst development when injected into both BCB+ and BCB- embryos.