159 Differential gene expression across bovine oocyte growth phase

Mammalian oocytes progressively acquire developmental competence during their growth phase, characterised by size-specific nuclear and cytoplasmic morphological changes and mRNA accumulation. Bovine oocytes express ∼12,000–15,000 transcripts, but the order of activation of their expression is unknown. We hypothesised that mRNA expression occurs sequentially rather than simultaneously during oocyte growth. To test this hypothesis, we aimed to characterise global mRNA expression in growing and fully grown bovine oocytes. Ovaries from Bos taurus animals were collected at a local slaughterhouse and sliced to recover oocytes from pre-antral and early antral follicles. The inside zona oocyte diameter was measured (60 to >120 μm), and individual oocytes (n = 81) were snap-frozen in RTL-Plus buffer. Single-cell RNAseq was performed and a total of 19,744 transcripts were identified. Principal component analysis (PCA) of global gene expression segregated the oocytes according to their size; oocytes of 60 to 79 (n = 29), 80 to 110 (n = 35), 110–119 and >120 (n = 19) μm in diameter clustered together. Hierarchical analysis segregated the highly expressed genes (1,134 genes above 0.3 standard deviation cut-off point) into two most significant clusters, where 497 genes presented a higher expression in growing oocytes (<110 μm) compared with oocytes greater than 110 μm in diameter. These genes were related to Wnt signalling pathway, DNA replication, and Golgi vesicle transport. On the other hand, 272 genes increased in expression across oocyte growth, including genes related to signal transduction, gene expression, and lipid metabolism. DESEqn 2 analysis was performed between groups that clustered separately in the PCA. A total of 3,547 genes were identified as differentially expressed genes (DEG) in oocytes < 80 μm compared with 80–110 μm (P < 0.05), highlighting genes related to transcription, negative regulation of mRNA catabolic process, microtube cytoskeleton organisation, and positive regulation of mitotic cell cycle. A greater number of DEG (6,346) were identified between oocytes of 80–110 to >110 μm. They were related to transcription, telomere maintenance, meiosis I, and positive regulation of histone modification. Similarly, 6,519 DEG were identified by comparing transcriptomes of oocytes < 80 μm and >110 μm in diameter. They were associated with different biological processes, including regulation of transcription initiation, negative regulation of intrinsic apoptotic signalling pathway, and autophagy. In conclusion, the present study demonstrates significant changes in the global gene expression profile of individual bovine oocytes as they progress through the growth phase. Moreover, gene ontology analysis revealed a higher metabolism and nuclear-cytoplasmic activity in growing oocytes compared with fully grown ones, which demonstrated activation of chromatin organisation and protein modification pathways.

This project is funded by the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 860960.