149 Molecular pathways associated with oocyte growth in cattle

In cattle, oocyte transcription starts during the secondary follicle stage, increasing in intensity in the early tertiary stage. Previously, cohorts of genes which either increased or decreased expression across the oocyte growth phase were identified by our group. The aim of the present study was to test our hypothesis that the oocyte transcriptome modulates during oogenesis to meet the changing requirements of the growing oocyte. To this end, Bos taurus oocytes were recovered from abattoir ovaries by slicing, their diameter was measured, and individual oocytes were snap-frozen in RTL-Plus buffer. A total of 179 oocytes were processed for single-cell genome and transcriptome sequencing; the resulting data were mined according to oocyte diameter: <70, 70–79, 80–99, 100–109, 110–119, and >120 μm. The data were mapped to the ARS-UCD1.2 bovine genome and analysed with RStudio (EdgeR) and ShinyGO 0.77 (gene ontology). Oocytes from secondary follicles (<70 μm in diameter) express genes associated with ribosome biogenesis (RPL5), mitochondrial function, and ATP production (COX2, ND1, ND5, ATP6, ATP8), whereas expression of maternal-effect genes (ZPs, GDF9, PADI6, BMP15, UHRF1, NLRP5, TLE6) was up-regulated in oocytes from multi-laminar preantral follicles (70–79 μm in diameter). In line with the high cytoplasmic activity of oocytes from early antral follicles (80–99 μm in diameter), the expression of genes related to regulation of transcription (DPPA3, CDC25B, NOTO), intracellular transport (CDC42, SCYL2, VPS41), and proteasome assembly (PSMG1) were increased. Reflecting oocyte acquisition of meiotic competence towards the end of the growth phase, nuclear pathways including meiotic nuclear division (WEE2, TRIP13, AURKA), chromosome segregation (KIF18A, CDC27, ATM, SMC4), and double-strand break repair (TDP2, NIPBL, KMT5B) were up-regulated, whereas those associated with cytoplasmic pathways were down-regulated in oocytes >100 μm in diameter. Bisulphite sequencing indicated stable global CpG methylation at ~15% in oocytes until the early antral follicle stage; thereafter, it increased up to >40% in fully grown ones (>120 μm). The highest increment in global methylation (10%) occurred at the end of the growth phase between 100–109 and 110–119 μm oocytes. This stage was also characterised by the greatest number of differentially expressed genes (4364) with up-regulation of histone lysine methyltransferases (KMT2E, KMT5B) and acetyltransferases (KAT6B, KATNBL1), and down-regulation of histone lysine demethylases (KDM5B, KDM5C) and deacetylases (HDAC1, HDAC11, HDAC6, HDAC7), as well as genes involved in DNA methylation (UHRF1, TRIM28, TET3) and the subcortical maternal complex (TLE6, NLRP2, NLRP5, PADI6). The expression of the DNA methyltransferases (DNMT3A, DNMT3B, DNMT1) remained stable during the oocyte growth phase, perhaps reflecting a steady-state turnover. In summary, the present study identifies key pathways associated with specific oocyte and follicle developmental stages. Our data indicate that an intensive pathway-specific program of transcription precedes de novo DNA methylation during the bovine oocyte growth, with both processes most active in the final stage of the growth phase.