279 CYTOPLASMIC POLYADENYLATION-REGULATED GENE EXPRESSION DURING BOVINE OOCYTE MATURATION

Abstract

Using RNA-seq of GV and MII oocytes we have described changes in polyadenylated transcript abundance that occur during in vitro bovine oocyte maturation (Reyes J. M., J. L. Chitwood, and P. J. Ross. 2013. Deciphering regulation of transcript abundance in maturing bovine oocytes. Poster session presented at International Plant & Animal Genome XXI. San Diego, CA). These changes can be attributed to transcript degradation, transcription, or transcript polyadenylation levels. The objectives of the present study were to determine the extent of cytoplasmic polyadenylation (CP) by measuring total and polyadenylated transcript abundance and poly(A) tail length in GV and MII oocytes for 8 (CCNB1, CPEB4, DNMT3B, FBXO43, EZH2, GDF9, PRDX2, and PAIP2) genes selected based on RNA-seq results. Oocytes were obtained by aspiration of abattoir-derived ovaries (GV) and in vitro maturation for 24 h (MII). Four pools of 40 oocytes were collected per stage. Enhanced green fluorescent protein (EGFP) cRNA was spiked into each sample before RNA extraction using the PicoPure RNA Isolation Kit. Extracted RNA was equally divided for cDNA synthesis using either random hexamers or anchored oligo(dT) primers to detect total and polyadenylated transcripts, respectively. Quantitative PCR (qPCR) of target genes, EGFP (exogenous control), and PPIA (endogenous control) was performed in duplicate for each replicate and gene. Relative transcript abundance was calculated using the 2^{[–ΔΔC(T)]} method and statistically analysed using the Student's t-test. Transcript poly(A) tail length was determined for all but 2 genes (DNMT3B and EZH2) at the GV and MII stages using rapid amplification of cDNA ends poly(A) test (RACE-PAT). Two replicates of GV (n = 100) and MII (n = 100) pairs were collected to perform RACE-PAT followed by fragment analysis on a Bioanalyzer DNA 1000 chip. Polyadenylated RNA abundance levels matched those of the RNA-seq study for 7/8 genes using both PPIA and EGFP to normalise qPCR data, demonstrating the validity of RNA-seq results. Furthermore, total transcript levels for 6/8 and 7/8 genes remained unchanged when normalized to an endogenous and exogenous control, respectively. Combined, the results suggest a significant role for CP considering changes in polyadenylated transcript levels occur without changes in total RNA abundance. Also, changes in transcript abundance corresponded to differences in poly(A) tail length at the specific stages as determined by RACE-PAT. In conclusion, CP is the predominant mechanism responsible for changes in transcript abundance during oocyte maturation at least for the majority of the examined genes, though more in-depth studies are required to determine the global extent of CP. This study improved the atlas of CP-regulated genes potentially providing researchers with critical knowledge to improve in silico tools that predict genes regulated by CP based on presence, position, and distribution of motifs within the 3′ untranslated region.