The changing landscape of immune cells in the fetal mouse testis

Fetal testis growth involves cell influx and extensive remodeling. Immediately after sex determination in mouse, macrophages enable normal cord formation and removal of inappropriately positioned cells. This study provides new information about macrophages and other immune cells after cord formation in fetal testes, including their density, distribution, and close cellular contacts. C57BL6J mouse testes from embryonic day (E) 13.5 to birth (post-natal day 0; PND0), were examined using immunofluorescence, immunohistochemistry, and RT-qPCR to identify macrophages (F4/80, CD206, MHCII), T cells (CD3), granulocytes/neutrophils (Ly6G), and germ cells (DDX4). F4/80⁺ cells were the most abundant, comprising 90% of CD45⁺ cells at E13.5 and declining to 65% at PND0. Changes in size, shape, and markers (CD206 and MHCII) documented during this interval align with the understanding that F4/80⁺ cells have different origins during embryonic life. CD3⁺ cells and F4/80^-/MHCII⁺ were absent to rare until PND0. Ly6G⁺ cells were scarce at E13.5 but increased robustly by PND0 to represent half of the CD45⁺ cells. These immunofluorescence data were in accord with transcript analysis, which showed that immune marker mRNAs increased with testis age. F4/80⁺ and Ly6G⁺ cells were frequently inside cords adjacent to germ cells at E13.5 and E15.5. F4/80⁺ cells were often in clusters next to other immune cells. Macrophages inside cords at E13.5 and E15.5 (F4/80^Hi/CD206⁺) were different from macrophages at PND0 (F4/80^Dim/CD206^-), indicating that they have distinct origins. This histological quantification coupled with transcript information identifies new cellular interactions for immune cells in fetal testis morphogenesis, and highlights new avenues for studies of their functional significance.