106 Heat stress alters oocyte genome-wide DNA methylation patterns revealed at single base resolution

There is a great amount of evidence indicating that the mammalian oocyte is susceptible to heat stress (HS). HS affects the series of cellular and molecular events triggered during oocyte growth compromising oocyte maturation, fertilization, and preimplantation embryonic development. DNA methylation reprogramming is a major event that takes place during oocyte growth and early embryo development. Changes in the epigenome may be induced by environmental exposures; however, little is known regarding the effects of heat stress during oogenesis. We have recently developed a new mouse model to target HS during the first wave of de novo DNA methylation that recapitulates several hallmarks of HS-mediated detrimental effects on oocyte developmental competence. Therefore, the objective of this study was to prospect the impact of HS on oocyte whole-genome bisulfite sequencing (WGBS) technology. To the best of our knowledge, there is no report describing genome-wide effects of HS on the oocyte epigenome. Lactating Swiss female mice together with Day 10 (postnatal day (P)10) postnatal litter pups were randomly allocated to HS (35°C for 12 h/light period and 21°C for 12 h/dark period) or control (21°C for 24 h) until weaning at postnatal Day 21 (P21). Weaned F₀ females were transferred to control conditions (21°C) until puberty. At postnatal Day 35 (P35) HS (n = 18) and control (n = 13) females received an intraperitoneal administration of 5.0 IU equine chorionic gonadotrophin (eCG; pregnant mare serum gonadotrophin) and cumulus-oocyte complexes were collected 46 h later. Germinal vesicle stage oocytes were denuded by gentle pipetting and three pools of 200 oocytes per group (N = 3 biological and technical replicates) were used for WGBS analysis. Oocyte DNA was extracted, converted by sodium bisulfite, and subjected to library preparation. The library was sequenced using next-generation sequencing and reads were mapped to the mouse reference genome. Differently methylated regions (DMR) were identified by the DSS method, and hypomethylated and hyper-methylated regions concern only the value of methDiff. Functional enrichment of DMR overlapping genes was performed using g:Profiler. A total of 104 191 methylated cytosines were mapped in mouse oocytes and 41 563 cytosines were differently methylated between groups. HS oocytes displayed 28 053 hypomethylated and 13 510 hypermethylated cytosines compared to control oocytes. Further, 3525 DMRs were mapped between groups, while HS oocytes displayed 2555 hypomethylated and 703 hyper-methylated DMRs compared to control oocytes. Functional analysis led to a major enrichment for biological processes (e.g. nutrient transport, growth and development), cellular components (e.g. membrane, cytoskeleton) and transcription factors (including members of the TGF-β signalling pathway). In conclusion, HS alters genome-wide DNA methylation patterns and reveals novel potential targets for mitigating HS-mediated effects on oocyte developmental competence.