School of Agriculture and Food Science, University College Dublin, Ireland1, Babraham Institute, United Kingdom2
Abstract Text: The development of a competent oocyte depends on morphological and molecular changes during its growth phase. In cattle, the morphological transitions of the follicle and oocyte have been described in detail. However, knowledge of the underlying molecular modifications remains incomplete. Previously we described the modulation of the oocyte transcriptome across bovine oocyte growth; our current objective was to characterise the establishment of the oocyte methylome. Oocytes were recovered from slaughterhouse-derived ovarian follicles by cortex-slicing. The diameter of single oocytes was measured and they were snap-frozen in RTL-Plus buffer. A total of 179 oocytes were allocated to the following diameter groups: < 60 µm, 60-69 µm, 70-79 µm, 80-89 µm, 90-99 µm, 100-109 µm, 110-119 µm, and >120 µm. Single-cell Bisulphite Conversion Sequencing was performed, and the data was mapped to the ARS-UCD2.1 bovine genome, with the quantification of 39,337,556 cytosines. Global CpG methylation remained low (< 11.5%) in oocytes until they reached 90-99 µm in diameter when the methylation levels increased to ~28%. Active gain in methylation continued until oocyte growth was completed ( >120 µm in diameter), at which stage methylation stabilised at >48%. The methylation data was quantified over gene bodies for further analysis. Following clean-up, a total of 18,327 genes were identified for which methylation quantification data was available in all groups. Setting the oocyte diameter group >120 µm as the control, genes with more than a 25% difference in methylation between diameter groups were identified and enriched pathways were determined. Most of the genes presented low to medium methylation levels in the smaller oocytes (< 60 to 80-89 µm) but were highly methylated in the control group (~12,000 genes). The genes were related to spindle organisation, cell division, chromosome segregation, protein and histone modification, and cytoplasmic microtubule organisation, among others. As oocyte diameter increased above 90 µm (90-99 µm, 100-110 µm, and 110-119 µm), the number of differentially methylated genes decreased compared to the control group (4,428 to 459 genes), with no enriched pathways assigned to them. However, higher methylation of OOSP2 and ZARL1 in the control oocytes is noteworthy. Next, we evaluated the methylation status of imprinted genes since their methylation will be preserved in future embryos. From a total of 32 identified imprinted genes, 13 were characterised by a continuous methylation gain from the smallest to the largest oocyte group, starting with less than 20% in oocytes < 60 µm until more than 70% methylation in oocytes >120 µm. A group of 6 imprinted genes presented a medium level of methylation (30 – 70%) in the largest oocytes ( >120 µm). Interestingly, the methylation levels of 13 imprinted genes remained low (< 30%), e.g., IGF2, indeed 6 of these genes presented less than 6% methylation, e.g. OOEP. In conclusion, here we present novel information on methylation dynamics during bovine oocyte growth. Our data reveals that methylation onset is later and occurs at different speeds and levels at imprinted genes in bovine compared to other species. This information provides a platform to better understand the epigenetic events during bovine oocyte growth and highlights a potential window of sensitivity when oocyte quality may be enhanced or compromise.
.jpeg.jpg "Lais Barbosa Latorraca, MS (she/her/hers) photo")