Figure 2: Effect of busulphan treatment on mouse testes: (A) Reduction in size of testes after busulphan treatment. (B) Loss in weight of testes after busulphan treatment.(C) Testicular sections of busulphan treated testis were immuno-stained using OCT-4 antibody to detect the presence of VSELs. Nuclear OCT-4 positive VSELs were detected in germ cells depleted, busulphan treated testicular sections.(D-F) Multiple fields showing nuclear OCT-4 positive VSELs (arrowheads) in busulphan treated testicular smears. H & E stained sections of (G) normal and (H) busulphan treated testes. Note the extensive germ cell depletion and collapsed Sertoli cells after busulphan treatment. Loss of germ cells after busulphan treatment was confirmed by immunolocalization for DAZL. Note the presence of DAZL positive germ cells (spermatogonia and early spermatocytes) in (I)normal testis which are lacking in (J) busulphan treated sections.(K) Differential expression of spermatogonial stem cell (Gfra) and germ cell (Dazl) specific transcripts in testis from control and busulphan treated mice. Note the marked reduction of Dazl (p<0.01) and Gfra (p<0.05)after busulphan treatment.(L)After busulphan treatment, the proportion of Oct-4A in total Oct-4 as indicated by the ratio of Oct-4A to Oct-4 is increased (p=0.101), indicating selective reduction of Oct-4B form. (M) Differential expression of VSELs specific marker Sca-1 transcripts in control and busulphan treated mice testes. Increased Sca-1 expression is observed after busulphan treatment (p= 0.28). Error bars represent standard deviation between at least three biological replicates. Bar = 20μm.