mKITL expression in Sertoli cells is critical for maintenance of Kit⁺ SPCs in testis. (A) Immunostaining for KIT (red), VE-cadherin (endothelium; green), β3-tubulin (Sertoli cells; blue), counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (nuclear stain; gray) in 4-week-old wild-type mouse testes. Representative of 12 sections from 3 separate mice. Arrow heads: KIT⁺ spermatogonial precursors adjacent to β3-tubulin⁺ Sertoli cells. Scale bar represents 50μm. (B) Immunostaining against cytoplasmic domain of mKITL (red), VE-cadherin (green), β3-tubulin (blue), counterstained with DAPI (gray) in 4-week-old AmhΔEx7 and wild-type mouse testes. Representative of 4 to 7 mice of each genotype. Scale bars represent 50μm. (C) Mean (± SEM) blood serum sKITL levels in 4-week old wild-type (control; n = 3) and AmhΔEx7 (n = 7) mice. (D) Immunostaining for KIT (red) and GFRA1 (green), counterstained with DAPI (gray) in 4-week-old wild-type control and AmhΔEx7 mouse testes. White arrows: KIT⁺ SPCs; yellow arrowhead: GFRA1⁺ spermatogonia. Representative of 15 mice of each genotype. Scale bars represent 50 μm. (E) Mean (± SEM) KIT⁺ SPCs and GFRA1⁺ cells per seminiferous tubule in 4-week-old wild-type (control; n = 19) and AmhΔEx7 (n = 30) testes (mean ± SEM). More than or equal to 50 round cross sections of seminiferous tubules were counted per genotype. The nonparametric Mann-Whitney test for multiple comparisons was used to assess statistical significance. ∗∗∗∗P < .0001. L, KIT⁺ Leydig cells; V, blood vessel.