Gpnmb-positive lipid-associated macrophages contribute to lipid clearance during luteolysis

Macrophages are important regulators of ovarian physiology, yet the complexity in their subpopulations and functions is far from fully understood and is emerging from single-cell analysis of diverse physiological and pathological contexts. One open question in ovarian physiology is how massive lipid loads during structural luteolysis are resolved without inducing inflammation. Here, by integrating single-cell and spatial transcriptomic datasets from mouse and human ovaries with high-resolution immunostaining and whole-mount imaging, we identified a Gpnmb+ subset of macrophages that dynamically expand during luteolysis in the mouse ovary, and a similar GPNMB+ macrophage population is also present in human ovarian scRNA-seq datasets. Transcriptomic profiling revealed that these Gpnmb+ macrophages resemble lipid-associated macrophages (LAMs) in adipose tissues in obesity, displaying strong enrichment in phagocytosis, lysosomal lipid processing, and reverse cholesterol transport. Notably, unlike phagocytic macrophages that only clear cellular debris, Gpnmb+ macrophages represent a distinct, specialized subset dedicated to processing lipid-rich targets: they selectively infiltrate regressing corpora lutea (CL), internalizing lipid droplets and interacting with dying luteal cells via apoptosis (Thbs1-Sdc1/4), efferocytosis (Pros1-Mertk), and cell recruitment (Cxcl12/Cxcr4) signaling axes. Gpnmb+ macrophages also surround oocytes in atretic follicles. Through integrated transcriptomic and imaging analyses, our data suggest that Gpnmb+ macrophages execute a complete cascade of multi-stepped lipid processing, including lipid droplet internalization, lysosomal and lipophagic degradation, and ABCA1/ABCG1-mediated cholesterol efflux. Furthermore, whole-mount imaging revealed close physical interaction between Gpnmb+ macrophages and LYVE1+ lymphatic endothelial cells, supporting a non-inflammatory resolution for lipid clearance via lymphatic circulation. Collectively, our findings establish Gpnmb+ macrophages as specialized LAMs essential for maintaining cyclic ovarian lipid homeostasis. This work redefines the paradigm of LAM biology by demonstrating their vital homeostatic role in normal ovarian physiology, bridging immune-mediated tissue remodeling and reproductive physiology.