RNA‐Seq profiling of single bovine oocyte transcript abundance and its modulation by cytoplasmic polyadenylation

Abstract

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, producing 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.

Cytoplasmic polyadenylation in mammalian oocyte maturation

“…One possibility is that alternate 3′UTRs are utilized for a subset of CCNB1 transcripts allowing for differential regulation of CCNB1 translation (Figure ). Other studies have reported (as do we) a shorter (~33 bp) variant of the bovine CCNB1 3′UTR with a Hex lacking upstream CPE that could explain the early translation of CCNB1 . Alternatively, looking at the sequencing reads mapping to the bovine genome, we found evidence of a third variant of approximately 3000 bp in length containing a multitude of regulatory elements including TCS and MBE (Figure ).…”

“…Complete 3′UTR (those containing a Hex within the last 30 bp) were examined for the presence, position, and distribution of CPE, PBE, and Hex sequences to determine potential cases of CPE‐mediated translational regulation, which we paralleled in human, mouse, and bovine MII oocytes (Table ). Of the 279 and 918 significantly (p‐value < .05 and fold change >2, DESeq) up‐ and downregulated genes with a complete 3′UTR, respectively, 85% of upregulated genes harbored regulatory motifs in line with translational regulation, whereas only 18% of downregulated genes were predicted to be regulated . Interestingly, we also found a tendency for enrichment of motifs within expressed genes at the MII stage in the three species, as suggested by the increased proportion of genes predicted to be regulated among expressed vs. total genes (Table ).…”

“…Another well‐characterized gene in terms of CP is CCNB1 , which produces a protein that together with CDK1 (aka cdc2) forms the MPF that drives oocyte maturation through its involvement with chromosome condensation, breakdown of the nuclear lamina, and cytoskeletal reorganization. In Xenopus, CCNB1 was determined to be regulated by CPE‐mediated CP, which was also confirmed in mice and presumably occurs in cattle and pig . However, the Xenopus 3′UTR structure greatly differs from MOS , containing a PBE, multiple CPEs, and a putative MBE.…”

“…Poly(A) tail length affects transcript translational efficiency, with short and long tails generally corresponding to repressed and active mRNAs, respectively, making measurement of polyadenylated transcript abundance useful in identifying changes in transcript utilization during oocyte maturation. Polyadenylated transcript abundance was recently examined by RNA‐seq in single GV ( n = 3) and MII ( n = 3) bovine oocytes revealing hundreds of candidate genes for translational regulation . Owing to the incomplete nature of the bovine genome, the annotation was extended and the longest 3′UTR extracted for each gene followed by combinatorial motif code analysis .…”

In search of the transcriptional blueprints of a competent oocyte

“…However, the new era of RNA sequencing aims to the single cell analysis thanks to an amplification step before library preparation, recent examples of which are available in cattle too (Chitwood et al, 2013;Reyes et al, 2015).…”