The epididymis comprises a single, highly coiled duct divided into initial segment, caput, corpus, and cauda, along which spermatozoa undergo progressive post-testicular maturation driven by region-specific epithelial secretory activity. The epithelium consists of principal cells, proton-secreting clear cells, basal cells with progenitor-like properties, and intraepithelial macrophages. Immunologically, the proximal and distal epididymis mount compartmentalized responses to infection, forming a characteristic inflammatory gradient along the organ. Flow cytometry and single-cell RNA sequencing identify macrophages as the dominant immune population within the tissue, positioning them as candidate regulators of this gradient. We therefore hypothesized that intraepithelial macrophages act as spatial regulators of epithelial homeostasis under both physiological and inflammatory conditions.
To interrogate macrophage–epithelial interactions, we achieved ~90% macrophage depletion throughout the epididymis for up to 30 days using the CSF1R inhibitor PLX5622. Immunofluorescence and morphometric analyses revealed a significant increase in tubule diameter (~10%) and basal cell (KRT5⁺) projection length (~30%) exclusively in the initial segment, while no histological alterations in the caput, corpus, or cauda were detected. This striking regional specificity implicates macrophages as non-redundant regulators of epithelial architecture in the most proximal epididymal segment, where luminal conditioning is critical for downstream sperm maturation.
To probe reciprocal epithelium-to-macrophage signaling, we employed a basal cell– specific CSF1 depletion model to destabilize the macrophage niche. This resulted in compensatory CCR2⁺monocyte-derived macrophage recruitment selectively to the cauda within 10 days, regulated by as of yet unknown signals.
Together, these findings reveal that epididymal macrophage homeostasis is regionally autonomous, that epithelial CSF1 is a non-redundant niche factor in the cauda, and that distinct macrophage subpopulations are functionally specialized along the organ axis. Given the relevant role of epididymitis in male infertility, these data open a new framework for understanding how macrophage–epithelial crosstalk safeguards the luminal environment permissive for sperm maturation.