Single-cell RNA sequencing uncovers cellular geterogeneity of granulosa cells and provides a signature for follicular development in chicken.

Functional differentiation of the granulosa cell (GC) layer plays an essential role in follicular development and oocyte maturation in birds. However, despite the importance of GCs, heterogeneity within the granulosa layer across poultry species remains poorly defined and functional transcriptomic signatures across distinct cellular subpopulations are lacking. In this study, single-cell RNA sequencing was used to identify GC types, uncover heterogeneity, and construct the developmental trajectories of chicken GCs at two developmental stages: the hierarchical follicle (HF)-GC and prehierarchical follicle (PHF)-GC stages. The following four GC types were identified: rapid growth, early, luteal, and primitive GCs. We found significant differences in the abundance of these different cell types at the HF-GC and PHF-GC stages. We also identified four potential differentiation trajectories for GCs during follicular development. The four distinct developmental trajectories may elucidate that the dynamic interplay and transition among these four GC types are pivotal in determining the fate of the follicle. A single-cell regulatory network inference and clustering analysis was performed to identify specifically expressed transcription factors in the different GC types. These transcription factors may have key regulatory roles in follicular development. Furthermore, we identified genes that were differentially expressed in HF-GCs and PHF-GCs. In total, 1,049 differentially expressed genes were identified in the two groups, including 379 upregulated and 670 downregulated genes. Finally, we identified several genes that may play important roles in follicle selection and development in chicken, including CDK2, CCND1, CCND2, BCL2, FOXO1, AMH, WT1, STAR, CYP11A1, HSD11B2, CYP51A1, and ESR1. These findings enhance our understanding of the cell biology of the GC layer in ovarian follicles‌ ‌and provide a basis for future studies of the relevant molecular regulatory mechanisms.