RNA-Seq profiling of single bovine oocyte transcript abundance and its modulation by cytoplasmic polyadenylation
Molecular Reproduction and Development
Molecular changes occurring during mammalian oocyte maturation are partly regulated by cytoplasmic polyadenylation (CP) and affect oocyte quality, yet the extent of CP activity during oocyte maturation remains unknown. Single bovine oocyte RNA sequencing (RNA-Seq) was performed to examine changes in transcript abundance during in vitro oocyte maturation in cattle. Polyadenylated RNA from individual germinal-vesicle and metaphase-II oocytes was amplified and processed for Illumina sequencing,
... ducing approximately 30 million reads per replicate for each sample type. A total of 10,494 genes were found to be expressed, of which 2,455 were differentially expressed (adjusted P<0.05 and fold change >2) between stages, with 503 and 1,952 genes respectively increasing and decreasing in abundance. Differentially expressed genes with complete 3'-untranslated-region sequence (279 increasing and 918 decreasing in polyadenylated transcript abundance) were examined for the presence, position, and distribution of motifs mediating CP, revealing enrichment (85%) and lack there of (18%) in up-and down-regulated genes, respectively. Examination of total and polyadenylated RNA abundance by quantitative PCR validated these RNA-Seq findings. The observed increases in polyadenylated transcript abundance within the RNA-Seq data are likely due to CP, providing novel insight into targeted transcripts and resultant differential gene expression profiles that contribute to oocyte maturation. Results Single oocyte transcriptome sequencing Single-oocyte, whole-cell lysates from GV (n=3) and MII (n=3) samples were used as template for cDNA synthesis, with an anchored oligo(dT) primer, and for amplification using a whole-transcript PCR amplification approach. Each stage yielded 21.7±2.8 ng (GV) and 20.9±4.7 ng (MII) of cDNA. Subsequent cDNA sonication, library preparation, and Reyes et al.