Single‐Cell RNA Sequencing Reveals the Role of Macrophage‐Granulosa Cell Crosstalk in Regulating Granulosa Cells Function in Sheep

Macrophages, as key immune cells in the ovarian stroma, play a pivotal role in regulating ovarian function and follicle development. To investigate the cellular dialogue between macrophages and granulosa cells and its role in the function of granulosa cells, we performed single-cell RNA sequencing on ovarian tissues from two contrasting breeds of sheep: the high-prolificacy Hu (HU) and the low-prolificacy Mongolian (MG) sheep. We identified 8 major cell clusters, including distinct macrophage and granulosa cell subsets. Comparative analysis showed breed-specific differences in the abundance of these two cell types, with HU ovaries containing more granulosa cells but fewer macrophages than MG ovaries. Integrative transcription-factor network analysis highlighted several regulators that differed between breeds, notably SOX4, FLI1, and FOXO1. In addition, we found the gene expression level of IGFBP2 in the HU group was significantly upregulated in granulosa cells and macrophages, compared with the MG group. Functional validation using an in vitro co-culture system demonstrated that knockdown of IGFBP2 in sheep macrophages significantly upregulated proliferation- and steroidogenesis-related gene expression and activation of the TGF-β pathway in granulosa cells, potentially through an IGF2-mediated mechanism. In conclusion, this study provides valuable insights into the intercellular communication between macrophages and granulosa cells in the ovarian microenvironment and provides novel insights into their regulatory mechanisms underlying reproductive capacity.