Figure 4. Depletion of macrophages leads to reduced cell cycle activity in undifferentiated spermatogonia.

(A–D) Tubule regions with a higher density of macrophages (A, B) contained more proliferative cells than those with fewer macrophages (C, D); magenta-colored cells are ZBTB16 (red)/BrdU (blue) double-positive cells (i.e., proliferative spermatogonia). Number indicates macrophages per 10,000 µm² of seminiferous tubule surface area within frame of image. Control samples had a greater percentage of MKI67-positive ZBTB16-positive cells (E’, arrowheads), whereas DT-treated samples had a higher percentage of MKI67-negative ZBTB16-positive cells (F’, arrows). DT-treated testes contained an increased percentage of quiescent CDKN1B–positive ZBTB16 cells (G’, H’, arrowheads) relative to non-quiescent ZBTB16 cells (G’, H’, arrows). E’-H’ are higher magnifications of the boxes in E-H, respectively. Scale bar, 50 εm. (I) Graph showing reduced numbers of proliferative cells (BrdU+) among all ZBTB16- positive cells, reduced active cell cycle status (MKI67+) within ZBTB16-bright cells, and increased quiescence (CDKN1B+) within ZBTB16-bright cells. Data are represented as mean percentage ± SEM. *, P<.05; **, P<.005. Panels A-D are from whole-mount seminiferous tubules; E-H are from cryosectioned testes. See also Figure S7.