Whole genome bisulfite sequencing of apoptotic and male-differentiated subpopulations of E13.5 wild-type murine male germ cells

Heterogeneity within murine prospermatogonia manifests as certain populations either die or survive during a developmentally scheduled apoptotic event at E13.5. We have identified that this apoptosis is clonal, suggesting that apoptotic fate is heritably enforced among populations of germ cells. To determine how subpopulations of germ cells differ, we used single-cell RNA sequencing to identify two diametric states: an apoptotic (AP) versus a male-differentiated (MD) subpopulation that differ primarily by the expression of critical male-differentiation genes known to be epigenetically regulated. We isolated AP and MD germ cells from E13.5 male embryos for whole genome bisulfite sequencing to compare how methylation differs between these populations. We find that AP germ cells are relatively hypermethylated at a whole-genome level and, importantly, are significantly hypermethylated at specific genome-reprogramming responsive (GRR) loci. Because these GRR genes facilitate male differentiation, we conclude that inappropriate demethylation as germ cells epigentically reprogram in the period preceding apoptosis produces an aberrantly male-differentiated clone that is fated for elimination. This quality-control paradigm therefore links fidelitous epigenetic reprogramming to germ cell selection. AP and MD germ cell populations were FACS-isolated from E13.5 male murine embryos from biological replicates. One replicate contained 2000 cells per each population while the second replicate contained 6000 cells per each population. Sequencing was completed for whole-genome analysis as well as specifically for 45 GRR genes and LINE1 elements.