Genome-wide maps of DNA methylome during oocyte aging and its transgenerational inheritance

The goals of this study are to compare genome-wide DNA methylation levels in young and aged oocytes,and to investigate the transgenerational inheritance of methylome profiles in oocytes during natural aging. We apply a novel protocol of rapamycin to overcome the DNA methylation drift associated with oocyte aging. 8-week-old female mice were injected intraperitoneally with rapamycin or vehicle for 40 weeks. At the end of the experiment, females (48 weeks, F0) were paired with young adult (~4 mo old) males to produce F1 offspring (OF1 and ORaF1). An F2 generation (OF2 and ORaF2) resulted from mating F1 female at 44~48 weeks of age with young adult males. To generate YF1 and YF2 as normal control (offspring of young mother), we mated females (~8 weeks) with young adult males. Then we collect oocytes (F0,F1 and F2 generations)，sperm ( F1 and F2 generations) and hippocampus (F1 female offspring) from different groups to investigate the transgenerational inheritance of DNA methylome profiles associated with oocyte aging by the single cell whole-genome methylation sequencing (sc-WGBS). We found that oocytes from aged mother exhibited increased DNA methylation levels in CpG sites. Maternal aging related methylome changes can be inherited transgenerationally though oocyte to the germ line of F1 and F2 offspring. The application of rapamycin during the course of oocyte aging could reverse these DNA methylation alterations, and it can ameliorate several neurobehavioral aging trails that were in observed in aged oocyte offspring. WGBS-seq on DNA from hippocampal tissue revealed a number of differentially methylated (P<0.05) genes in OF1 and ORaF1 compared with YF1, and some of the enriched pathways were associated with aging process, such as PI3K-Akt signaling pathway (akin to transcriptional alterations above), MAPK signaling and Ras signaling pathway . Total 47 samples from F0, F1 and F2 generations. 9 samples from F0 mother(4-week-old as young control, 48-week-old and 48-week-old rapa-treated oocytes), 7 samples from F1 oocytes (YF1,oocytes from young mother; OF1,oocytes from aged mother; ORaF1,oocytes from rapa-treated aged mother), 7 samples from F2 oocytes (YF2, oocytes from YF1; OF2, oocytes from OF1; ORaF2, oocytes form ORaF1), 6 samples from F1 sperm( OF1,sperm from aged mother; ORaF1,sperm from rapa-treated aged mother), 9 samples from F2 sperm( YF2, sperm from YF1; OF2, sperm from OF1; ORaF2, sperm form ORaF1),each group consists of 3 separate experiments, each sample contained about 15 oocytes. 9 hippocampus samples from F1 famale offspring(YF1, hippocampus from young mother; OF1, hippocampus from aged mother;ORaF1, hippocampus from rapa-treated mother), each group consists of 3 separate hippocampus from different offspring.