Single-cell RNA sequencing reveals oocyte-granulosa crosstalk and regulatory networks driving chicken primordial follicle assembly.

Primordial follicle assembly is crucial for ovarian development and reproductive capacity in chickens, yet the regulatory mechanisms remain unclear. Using single-cell RNA sequencing, we characterized germ cell-granulosa cell interactions during key ovarian developmental stages. Here, we present a single-cell transcriptomic atlas of chicken germ cells and granulosa cells at single-cell resolution. Our analysis revealed significant cellular heterogeneity among both germ cells and granulosa cells during primordial follicle assembly. Germ cells were categorized into six subtypes: mitotic S, mitotic G2/M, pre-meiosis, meiosis, oocyte, and dying oocyte by using UMAP analysis. Results revealed that germ cell fate determination is governed by stage-specific genes (KIF11, C14ORF39, LHX8) and transcription factors (MCM5, SMC1B, GDF9, SQSTM1), ensuring meiotic fidelity and high-quality primordial follicle formation. Pseudotime trajectory analysis demonstrated that oocytes undergo autophagy and apoptosis under bidirectional regulation by metabolic and stress-responsive pathways. Four granulosa cell subtypes (PreGC1-4) were identified, with roles in: steroid synthesis (CYP17A1, STAR), extracellular matrix formation (COL1A1, COL3A1), proliferation regulation (PCNA, MCM3), and cellular communication (NTN1). CellChat analysis enhanced crosstalk via WNT, ACTIVIN, BMP pathways, safeguarding structural and functional follicle integrity. Notably, the WNT4/β-catenin pathway acts as a central regulator during the late stage of assembly by activating granulosa cell function. It drives proliferation via CDK2 upregulation and guides differentiation through the transcription factor WT1, thereby promoting follicular formation. This study provides a molecular framework for chicken primordial follicle assembly, identifying key genes/pathways that lay a foundation for optimizing poultry reproductive efficiency.