Figure 3.

Proliferation status of spermatogonia and Sertoli cells in testicular fragments cultured for 5 weeks. (a) Double immunohistochemical staining of MAGEA4 and PCNA. Proliferative spermatogonia were identified by simultaneous expression of MAGEA4 and PCNA while proliferative Sertoli cells (SCs) were morphologically identified. Black arrow, PCNA-negative spermatogonia; white arrow, PCNA-positive spermatogonia; black arrowhead, PCNA-negative SCs; white arrowhead, PCNA-positive SCs. Scale bar = 10 μm. Percentage of seminiferous tubules containing at least one proliferative (b) spermatogonia or (c) SCs in fresh (pink) and cryopreserved (blue) testicular fragments cultured in medium without (medium I; faint colour) or with FSH/LH supplementation (medium II; dark colour). Single dots represent results from individual patients, and the boxplot displays the median and 25th and 75th percentiles of all patients at each time point in culture. Mixed-effect linear model revealed a statistically significant reduction (P ≤ 0.001) during culture.