Figure 3

Metabolomic analysis of the culture medium and evaluation of new culture medium, Rat medium 2. (a) Summary graph showing changes in the concentrations of 23 substances, in the medium used for culturing P7 rat testis tissues, relative to those on day 0. Substances were classified into 3 groups according to a statistical analysis using Jonckheere-Terpstra test: substances showing increasing tendency (produced, marked by *), decreasing tendency (consumed, marked by ^#), and the rest (stable). (b) Changes in the measured concentration of seven substances categorized into the consumed group are shown. P values by the Jonckheere-Terpstra test were 0.004 for Asp, Pyruvate and Ile, 0.01 for Glucose, Glue and Val and 0.02 for Leu. (c) P5 Acr-EGFP rat testis tissue cultured with either rat medium 1 or rat medium 2 for 31 days. Both showed broad GFP expression. Bar graph shows GFP expression area ratio cultured either with rat medium 1 or 2. Five rats aged P5 to P9 were used and the tissues were cultured for 36 to 45 days. Tissue number; n = 22 (rat medium 1) and 14 (rat medium 2). BF stands for brightfield. (d) PAS-stained histological view of P7 rat testis tissues cultured for 41 days. Yellow arrowheads indicate elongating spermatids. (e) Band graph showing the ratios of seminiferous tubules classified according to the most differentiated germ cell type contained. The round spermatids in steps 1–4 have dot-shaped acrosomes, while those of steps 5–6 have cap-shaped acrosomes. Bar graphs show average ratio and standard deviation of tubules containing haploids (left) or elongating spermatid (right) in total number of tubules observed. Six rats aged P5 to P9 were used and the tissues were cultured for 36 to 45 days. Tissue number; n = 14 (rat medium 1) and 9 (rat medium 2). Scale bars: 2 mm (c), 50 µm (d top), 10 µm (d bottom).