DHX36 plays a crucial role in regulating transcriptional and post-transcriptional processes through its interaction with G-quadruplexes (G4s). The mechanisms by which DHX36 regulates G4s vary across different cell types and physiological conditions. Oocyte-specific conditional knockout (CKO) mice were utilized to study the impact of DHX36 deficiency on female fertility. The results show that the CKO mice exhibit severely impaired hormone response, ovulation, and complete infertility. The CKO germinal vesicle (GV) oocytes display large nucleoli, aberrant chromatin configuration, decreased chromatin accessibility, disturbed transcriptome, and inhibited meiosis progression. Following fertilization, the embryos derived from the CKO oocytes arrest at the zygote or 2-cell stage. Notably, we observed inadequate rRNA transcription in growing GV oocytes, as well as insufficient pre-rRNA processing and translation activity in fully-grown GV oocytes. Using a G4 probe and antibody, we found increased G4s formation at the chromatin and cytoplasm of CKO GV oocytes
these G4s mainly originate from the rDNA and pre-rRNA. Furthermore, the distribution of DHX36 was found to be spatiotemporally synchronized with that of pre-rRNA and G4s in early mouse embryos. In vitro experiments confirmed that DHX36 directly binds with pre-rRNA through the RHAU-specific motif (RSM). Overexpression of DHX36 could partially alleviate the pre-rRNA accumulation in fully-grown CKO oocytes. In conclusion, this study highlights the physiological significance of DHX36 in maintaining female fertility, underscoring its critical role in rRNA homeostasis and chromatin configuration through G4-unwinding mechanism in mouse oocytes.