RNase H1 surveillance axis coupling chromatin compaction
R-loops, though implicated in genome stability, have poorly defined transcriptional roles due to confounding replication processes in proliferating cells. Leveraging replication-free oocytes, we applied high-specificity R-loop mapping strategy (RTACC-seq) and identified dynamics R-loop enrichment not only at gene promoters, but also at distal transposable elements (TEs). Here we found that TE-associated R-loops prominently accumulated during the transcriptionally active stage and were efficiently resolved in fully grown oocytes (FGOs), coinciding with chromatin condensation and transcriptional silencing. Crucially, we demonstrated that R-loops exert opposing, location dependent regulatory functions suppressing transcription at promoters while enhancing it at TEs, where they function as distal enhancer-like elements to promote chromatin accessibility and transcriptional activation. Oocyte specific deletion of Rnaseh1, encoding an RNA,DNA hybrid specific endonuclease, led to persistent R-loops, chromatin decondensation, and impaired transcriptional silencing in FGOs, culminating in premature ovarian failure and female subfertility. These findings characterize the mechanistic basis of R-loop as a critical mediator that involves in chromatin remodeling, transcriptional activity, and germ cell developmental competence.