Fig. 7: In vivo transplantation of SCF-soaked/CM-DiI-labeled beads into the testicular interstitium induces proliferation of differentiating spermatogonia in Cldn11^−/− mice.

a Immunohistochemistry of testis sections prepared from busulfan-treated Cldn11^+/− and Cldn11^−/− mice using anti-SCF and anti-ZO1 antibodies. b Schema for in vivo transplantation of SCF-soaked/CM-DiI-labeled beads into the interstitium of Cldn11^−/− mouse testes. Appearance of a SCF-soaked/CM-DiI-labeled bead is shown. CM-DiI labels seminiferous tubules in proximity to the beads. c Whole-mount immunofluorescence staining using anti-KIT antibody on seminiferous tubules from Cldn11^−/− mouse testes, with bovine serum albumin (BSA)- or SCF-soaked beads transplanted into the interstitium. Seminiferous tubules with or without CM-DiI labeling are shown as CM-DiI⁺ and CM-DiI^-, respectively. White dotted lines outline the seminiferous tubules. d BSA- and SCF-soaked beads were separately transplanted into distinct testes of a Cldn11^−/− mouse. Transplantation was performed using six biologically independent Cldn11^−/− mice. After, 67 and 72 seminiferous tubules labeled with CM-DiI derived from BSA- or SCF-soaked beads, respectively, were analyzed. The number of KIT⁺ cells per 100 μm seminiferous tubule labeled with CM-DiI was quantified. Red dots indicate biological replicates of mice. Data are shown as mean ± SD and were analyzed by Welch’s t-test. e Working model showing the role of CLDN11 in maintenance of differentiating spermatogonia. CLDN11-based Sertoli cell tight junctions (SCTJs) physically divide seminiferous tubules into adluminal and basal compartments. KIT-positive differentiating spermatogonia are located at the basal compartment. Interaction of Sertoli cell-derived SCF with KIT is essential for survival of differentiating spermatogonia. SCF accumulates at the basal compartment through Sertoli cell polarization after SCTJ formation, presumably promoting KIT activation in differentiating spermatogonia. Cldn11 knockout delocalizes SCF from the basal compartment and reduces the number of differentiating spermatogonia, likely due to a decrease in KIT activity. Scale bars: 10 μm (a), 100 μm (b), and 50 μm (c).