Figure 1.
Sertoli cell-specific ablation in adulthood.
(A) Amh-Cre;iDTR model. (B) DTR expression is restricted to Sertoli cells (scale bar: 50 µm). (C) Schematic representation of experiments in this study showing the time points of ablation relative to testis development. DTX-induced testicular atrophy (D) 7 days, (E) 30 days or (F) 1 year after injection in adulthood (scale bar: 500 m). (G) Immunolocalisation for CLEAVED CASPASE3 shows that apoptosis is restricted to Sertoli and germ cells (scale bar: 50 µm). (H) SOX9 expression is reduced but is still present in occasional tubules 7 days post-Sertoli cell ablation (arrowhead). However, SOX9 is completely absent 30 days post-Sertoli cell ablation (scale bar: 100 µm). (I) Sertoli cell ablation is mirrored by a decrease in Sertoli cell specific transcript expression: Dhh, Fshr and Wt1 (one-way ANOVA n = 5–9, ***P<0.001). (J) Circulating FSH concentrations at d80 (one-way ANOVA n = 9–12, *P<0.05, **P<0.01). d,days; yr,year; dtx, injected with toxin; veh: vehicle control.
Figure 2.
Germ cell loss after Sertoli cell ablation in adulthood does not impact testicular architecture.
(A) Testis weight is decreased at all ages following Sertoli cell ablation in adulthood (T-test n = 8–12, ****P<0.0001). (B) Immunolocalization of DDX4 shows loss of Germ cells is complete 30 days following Sertoli cell ablation (scale bar: 100 µm). (C) Testicular histology (2.5 µm resin sections stained with hematoxylin and eosin) shows that intact tubules, containing spermatogonia, spermatocytes and spermatids, are present 7 days following Sertoli cell ablation although many of the spermatocytes (red arrowhead) are undergoing apoptosis. By 30 days the lumen of most tubules are either acellular or contain only elongated spermatids (black arrow). Low and high power pictures from treated animals are shown at each age. (D) Image on the right shows that one year after ablation tubular architecture is retained although the seminiferous tubules contain only calcium deposits and some elongated spermatids. The image on the left is a from a control, age-matched testis. (E) Expression of Germ cell markers Stra8 and Dkkl1 and Ptm2 (one-way ANOVA n = 5–9, **P<0.05 ***P<0.001 ****P<0.0001). (F) Consistent with Germ cell loss, Cauda epididymides are devoid of spermatozoa and only cellular debris remains 30 days post Sertoli cell ablation (arrowhead) (scale bar: 100 µm). d,days; yr,year; dtx, injected with toxin; veh: vehicle control.
Figure 3.
Germ cell-specific cell ablation in adulthood does not impact testicular architecture.
Injection of DTX at pnd50 in Stra8-Cre+/−;DTR+/− mice induces specific Germ cell ablation (DDX4 immunolocalization). Testis architecture is unaffected and key testicular somatic cells remain present - Sertoli cell (SOX9), PTMC (SMA), and Leydig cell (HSD3B) (scale bar: 100 µm).
Figure 4.
Sertoli Cells regulate PTMC function in adulthood.
(A) Testicular histology (2.5 µm resin sections stained with hematoxylin and eosin) shows a single layer of PTMC is retained around seminiferous tubules (red arrowhead) 30 days after Sertoli cell ablation at pnd50 although some disruption of the PTMC layer can be noted (red arrows, associated with a small number of macrophage-infiltrated tubules) (scale bar: 100 µm). (B) Expression of PTMC markers Cnn1 and Myh11 (one-way ANOVA n = 5–9, **P<0.05, ***P<0.001). (C) SMA, Calponin (functional marker), laminin (basement membrane) and MYH11 expression is retained 90 days following Sertoli cell ablation at pnd50. Disruption of SMA, Calponin, laminin and MYH11 is observed one year following ablation (scale bar: 100 µm), although they remain unaffected in testicular vasculature (inset). (D) Consistent with loss of PTMC function a fibrotic phenotype (PSR staining) develops one year following Sertoli cell-ablation (scale bar: 100 µm). d,days; yr,year; dtx, injected with toxin; veh: vehicle control.
Figure 5.
The PTMC/basement membrane inhibits immune cell infiltration of seminiferous tubules.
(A) Biotin is prevented from entering the adluminal compartment by the Blood-Testis-Barrier in control animals. Conversely, 30 days post Sertoli cell ablation at pnd50, and despite the continued presence of the PTMC layer, biotin penetrates throughout the intratubular space. MgCl2: vehicle control. (B) Immunolocalization of macrophages (GFP) at 30 days following ablation at pnd50 using Amh-Cre+/+;iDTR+/+;CSFR-GFP mice (GFP antibody) shows that very few tubules are infiltrated by macrophages (red arrowhead). Macrophages are mainly present in the interstitium (arrow) or surrounding tubules (white arrowhead). (C) Sections from resinembeded testis highlighting the rare intra-tubular macrophage infiltration (asterisks). (D) Relative expression of the inflammatory cytokine Tnfa at key ages following Sertoli cell ablation at pnd50 (one-way ANOVA n = 5–9, **P<0.01) (scale bar: 100 µm). d,days; yr,year; dtx, injected with toxin; veh: vehicle control.
Figure 6.
Sertoli cell ablation in adulthood induces apoptotic loss of Leydig cells.
(A) Testicular histology on 2.5 µm resin sections (stained with hematoxylin and eosin) shows altered Leydig cell morphology 30 days after Sertoli cell ablation at pnd50. Leydig cells show increased vacuolization of the cytoplasm (red arrowhead), while some cells have an altered nuclear shape (red arrow) or reduced cytoplasmic volume after Sertoli cell ablation (white arrowhead). (B) Immunolocalisation for CLEAVED CASPASE3 shows Leydig cells undergo apoptosis (red arrowhead) (scale bar: 25 µm). (C) Total Leydig cell number per testis at key time-points following Sertoli cell ablation at pnd50 (one-way ANOVA n = 3–13, ****P<0.0001). (D) Leydig cells (HSD3B) are restricted to the rete (red arrowhead) and subcapsular region (red arrow) following Sertoli cell ablation at pnd50. (scale bar: 100 µm). d,days; yr,year; dtx, injected with toxin; veh: vehicle control.
Figure 7.
Adult Leydig Cell functionally compensate following Sertoli cell ablation in adulthood.
(A) Serum testosterone (one-way ANOVA n = 7–14) (B) Seminal Vesicle weight (T-test n = 8–12) and (C) Luteinizing Hormone concentrations (T-test n = n = 7–14, **P<0.01) in adulthood following Sertoli cell ablation at different ages. d,days; yr,year; dtx, injected with toxin; veh: vehicle control; SV:seminal vesicle.
Figure 8.
Changes in hormone pulsatility following SC ablation.
Individual values for serum testosterone in vehicle-treated (A) and DTX-treated (C) animals. Individual values for serum LH in vehicle treated (B) and DTX treated (D) animals. Red dotted line indicates the mean of control animals. d,days; yr,year; dtx, injected with toxin; veh: vehicle control.