1 Microinjection of CPE-Binding Protein Polyadenylated mRNA Increases Developmental Competence of Bovine Oocytes In Vitro

Abstract

Developmental competence is acquired during oocyte growth and maturation while oocytes undergo both nuclear and cytoplasmic changes. Completion of oocyte maturation and subsequent embryo development relies mostly on maternally synthesised and stored mRNAs at the transcriptionally quiescent phase. The temporal and spatial post-transcriptional and translational regulation of the stored mRNA in mammalian oocyte cytoplasm is essential for developmental competence of oocytes and is often controlled via cytoplasmic polyadenylation. Cytoplasmic polyadenylation element (CPE)-binding protein (CPEB) is required for polyadenylation of most mRNAs during oocyte maturation. It has been reported that in vitro-matured oocytes with high developmental competence showed an increased level of CPEB mRNA in oocyte cytoplasm. Thus, we hypothesise that the introduction of exogenous CPEB mRNA into in vitro-matured oocytes could increase their developmental capability. In this study, we first synthesised polyadenylated CPEB mRNAs by in vitro transcription. Cumulus-oocyte complexes were recovered from slaughterhouse ovaries and subjected to in vitro maturation for 21 h. After the removal of cumulus cells, matured oocytes were parthenogenetically activated (5 min in 5 mM ionomycin followed by 4 h in 2 mM DMAP with 5 mg mL⁻¹ cycloheximide). Each activated oocyte was injected with 5 to 10 pL of poly(A)-RNA solution (400 ng μL⁻¹; CPEB mRNA and green fluorescent protein (GFP) mRNA for the injection group or GFP mRNA for the control group) using a micromanipulator. After injection, the oocytes were cultured in SOF medium supplemented with amino acids for 8 days. No difference was observed in cleavage rate between CPEB and control group. However, the blastocyst rate was significantly higher in the CPEB group than in the control (24.9 ± 2.9% v. 15.0 ± 4.5%; P < 0.05). Cleavage and blastocyst rates were analysed by one-way ANOVA. We also compared the gene expression profile of blastocysts derived from both groups. The blastocysts were collected individually and analysed by single-embryo RT-PCR. Twenty-two genes were selected for analysis based on their roles in genomic reprogramming and embryonic development and fell into 6 functional categories: growth regulatory factors, cell cycle regulation, imprinting, apoptosis, pluripotency and DNA methyltransferase. The single-embryo RT-PCR was performed using the Flex-Six integrated fluidic chip (Fluidigm Corp., South San Francsisco, CA, USA) on the BioMark platform (Fluidigm Corp.). Relative expression values were calculated using the ΔΔCT (fold change) method and analysed by ANOVA. We found that 6 genes (H19, GRB10, DNMT1, CCNB1, CDK2, and SOX2) were up-regulated and 3 were down-regulated (DNMT2, BAX, and P53), along with the overexpression of CPEB gene (P < 0.05). Our results demonstrate that developmental competence can be improved by injecting exogenous CPEB mRNA into in vitro-matured metaphase II cattle oocytes, which reaffirms the essential role of CPEB in early embryonic development.