Single-cell multi-omics analysis decodes molecular characteristics of sheep oocyte fate in vivo maturation

A competent oocyte in mammals undergoes a remarkably elaborate process during follicle growth. Oocytes in antral follicles of varying sizes are subject to the potential outcomes of maturation. However, the comprehensive multi-molecular mechanisms underlying oocyte development fate during antral folliculogenesis remain largely elusive. Here, we employed sheep, a large non-primate species of economic importance, as the study model. Oocytes and granulosa cells from antral follicles of varying sizes were collected and then analyzed with our modified single-cell multi-omics sequencing technology to profile their transcriptomes, DNA methylation patterns, and chromatin accessibility features. The in vivo matured metaphase II (MII) oocytes were utilized to infer the fate trajectories of different oocytes. Transcriptomic profiling unveiled three distinct oocyte subtypes (Type 1, 2 and 3), with Type 3 oocytes identified as those progressing towards a maturation-competent fate. DNA methylation and chromatin accessibility data also revealed distinct differences among those three oocyte types. A detailed analysis of Type 3 oocytes revealed that, while preparing key factors for maternal RNA degradation (M-decay), those oocytes accomplished the majority of maternal RNA clearance. Moreover, cell-cell communication analysis between oocytes and granulosa cells revealed a high enrichment of the Endothelin 1 (ET-1) signaling pathway in Type 3 oocytes. Further experimental validation showed that adding ET-1 enhanced the function of important organelles, such as mitochondria, endoplasmic reticulum, Golgi apparatus, and cortical granules in mature oocytes, facilitating spindle assembly and thereby improving oocyte developmental competence. Conversely, ET-1 receptor antagonists (BQ123) led to a decline in oocyte quality. Notably, ET-1-treated oocytes exhibited elevated blastocyst formation rates post in vitro fertilization (IVF). In summary, our study provides new insights into the orchestration mechanisms governing oocyte fate determination in mammals and offers a novel strategy for enhancing sheep oocyte quality and developmental potential during in vitro maturation.