Figure 3

Progression of spermatogenesis following in vitro maturation of mouse testicular tissues treated by chemotherapy. (a) Microscopy images showing the general appearance of testicular tissue exposed to monochemotherapy (VCR or CYP), polychemotherapy (VCR+CYP) or unexposed (control) following 30 days of in vitro maturation. The necrotic area in cultured tissues has been delimited by dotted lines. (b) TRA98 immunostaining associated with Periodic Acid Schiff reaction was performed to visualize the progression of spermatogenesis after 30 days of organotypic culture, by detecting TRA98+ germ cells (spermatogonia, leptotene/zygotene and pachytene spermatocytes) and the pink-labelled acrosome (PAS+) of spermatids in control, CYP, VCR and VCR+CYP-treated mice. Intratubular cells were identified as: S for Sertoli cells (irregular TRA98- blue nuclei close to the basement membrane), Sg for spermatogonia (smooth spherical TRA98+ brown nuclei close to the basement membrane), L/Z for leptotene/zygotene spermatocytes (irregular spherical TRA98+ brown nuclei with condensed chromatin), P for pachytene spermatocytes (irregular spherical TRA98+ brown nuclei with highly condensed chromatin), RS for round spermatids (regular small round TRA98- blue nuclei and PAS+ acrosomal cap) or ES for elongated spermatids (elongated TRA98- blue nuclei with highly condensed chromatin and PAS+ acrosomal cap). Panels on the lower right corner represent a higher magnification of elongated spermatids (). (c) Percentage of seminiferous tubules at the most advanced differentiation stage of spermatogenesis after in vitro maturation of testicular tissues from mice exposed to VCR, CYP, VCR+CYP or unexposed (control) during the prepubertal period. Data are presented as means ± SEM with n = 4 testicular explants for each group. A value of *P < 0.05 was considered statistically significant.