Figure 3. Ablation of macrophages results in decreased numbers of spermatogonia.

(A) Timeline of the multiple-DT-injection scheme. Each blue bracket represents one day; macrophage depletion period is shown by a black line. DT injections were administered on days 1, 3, 5, and 7. Testes were harvested 24-72 hours after last injection. The mouse spermatogenic cycle is depicted, from A-type undifferentiated spermatogonia (A_undiff) to intermediate (mitosis) differentiated spermatogonia (In_m). Expression of stage-specific spermatogonial markers is shown below. (B) Control PBS-injected tubule. (C) “DT a” tubule. Group “DT a” denotes a minority of tubules (<10%) containing small numbers of macrophages. (D) “DT b” tubule. Group “DT b” denotes the majority of tubules (>90%) completely devoid of macrophages. SOX9 labels Sertoli cells. (E–G) Graphs show extent of macrophage ablation (E), Sertoli cell number (F), and total ZBTB16, ZBTB16-bright and ZBTB16-dim cell number (G). Data are represented as mean ± SEM. *, P<0.05; **, P<0.005. (H, I) Control Cre-negative samples had a greater percentage of CDH1-positive long A_aligned chains (H, arrowheads), whereas macrophage-depleted samples were biased towards shorter chains (H, I, arrows). (J, K) Macrophage-depleted testes showed a reduction in the basal-most population negative for DDX4 (bracket in J’); VCAM1 labels myoid cells overlying seminiferous tubules and labels interstitial cells. J’ and K’ are higher magnifications of the boxed regions in J and K, respectively. Scale bar, 50 mm. Panels B-D and H-I are from whole-mount seminiferous tubules; J-K are from cryosectioned testes. Graph in L shows numbers of different spermatogonial types (as defined by interconnected CDH1-expressing cells) per unit area. Data are represented as mean ± SEM. *, P<0.01. See also Figures S4–S6.