Figure 2
From: Generation of rat offspring using spermatids produced through in vitro spermatogenesis
In vitro spermatogenesis in the cultured Acr-EGFP rat testis tissues. (a) Stereomicroscopic view of P8 rat testis tissues from culture day 0 to 34, using a PC chip with dent depth of 150 μm under 15% O2. Brightfield (BF) and GFP fluorescence (GFP) pictures were shown. (b) Stereomicroscopic view of P7 rat testis tissues cultured for 49 days, without and with a PC chip covering, left and center & right, respectively. The dent depth in the PC chip was 160 μm. Arrowheads indicate strong GFP aggregation in the acrosome in spermatids. (c) Histological section of a cultured tissue stained with PAS showed round spermatids having a PAS-stained acrosomal cap (yellow arrowheads). P7 rat testis tissue cultured for 43 days under a PC chip. The dashed rectangular area is enlarged in the bottom panel. (d) Stereomicroscopic view of P3 rat testis tissues cultured for 44 days; BF and GFP fluorescence pictures with a 160 µm-PC chip covering are shown. (e) Cryosection of the cultured tissue shown in (d) was stained with anti-GFP antibody (green), PNA for acrosome (red), and hoechst 33,342 for nuclear (white). The dashed rectangular area is enlarged in the bottom panel. (f) Flowcytometry of a dissociated cell suspension derived from testis tissue directly from an adult Acr-EGFP rat at 5 months of age (in vivo) and from the tissue of a P7 rat cultured for 50 days with a PC chip covering (in vitro). Numbers beside each area enclosed by a colored line represent the proportion of enclosed cells to total cells. R4: 1n; R5: 2n; R8: 4n. (g) Comparison of the haploid cell ratio in total cells and number of haploid cells per tissue between tissue samples treated with and those treated without a PC covering. Red and blue crossbars in the box indicate the median. Black crossbars indicate the average. P7–8 rat testis tissues were cultured for 49–55 days. The number of PC (−) tissues analyzed was 5 from 5 animals, and the number of PC (+) tissues analyzed was 8 from 4 animals. Scale bars: 1 mm (a), 500 μm (b, d), 50 μm (c upper), 10 μm (c bottom), 20 μm (e).
