Macrophages promote survival of granulosa cells

Abstract The ovary contains numerous macrophages, and macrophage depletion in mice leads to dysfunctional ovulation. Macrophage function in the ovary is presumed to be related efferocytosis, however, the absence of macrophages also disrupts follicular development and corpus luteum development. Granulosa cells (GC) are critical to ovarian follicle development and survival, and we hypothesized that macrophage-GC crosstalk promoted GC function. To determine whether macrophage-GC interaction has an anti-apoptotic effect on GCs, BMDM were polarized to proinflammatory phenotype with LPS (M1), alternative activation phenotype with IL-4 (M2), or not stimulated (M0). After removing stimuli, primary mouse GCs were co-cultured with macrophages and serum-starvation was used to induce apoptosis of GCs. The cells were analyzed by flow cytometry, assessing viability by exclusion of propidium iodide and Annexin V. Co-culture with macrophages protected GCs from serum-starvation induced cell death. In co-cultures with quiescent BMDM, 60% of GCs were late apoptotic/early necrotic compared to 81% without co-culture, (p=0.0031). Unexpectedly, LPS-treated macrophages promoted survival of GC slightly more effectively than IL-4-treated or quiescent macrophages (45% versus 60% and 53% apoptotic, respectively p=0.0230). Both quiescent and inflammatory macrophages promote survival of GCs. This result is unexpected, as LPS-treated macrophages secrete TNF-α, a cytokine that is known to induce death in GCs. This study elucidates the role of macrophages in ovarian function by supporting GC survival and may also have implications for the promotion of GC tumors by tumor-associated macrophages. Supported by grant from the NIH 3P30GM118228-05S3