Figure 1

Preparation of ALCs from XY and XX/Sry testes. (a) XY and XX/Sry testes from eight-week-old Ad4BP-BAC-EGFP/mFLE-mCherry mice were immunostained with antibodies for EGFP (green), mCherry (red), and HSD3B6 (white). Nuclei were stained with DAPI (blue). Merged images are shown on the left. Arrowheads indicate FLCs (EGFP/mCherry double-positive, HSD3B6-negative). ALCs were detected as EGFP/HSD3B6 double-positive, mCherry-negative cells. Scale bars = 20 μm. (b) Whole cell preparations from the testes of 8-week-old XY and XX/Sry mice were analyzed via FACS. The cells surrounded by solid lines were recovered as EGFP single-positive ALCs, whereas those surrounded by dotted lines were recovered as EGFP/mCherry double-positive FLCs (upper panels). Total testicular cells were counted. Numbers of ALCs and FLCs per testis are calculated using the results obtained by FACS. Nine biologically independent samples (n = 9) were used for counting (lower panels). (c) The expression levels of marker genes in the sorted XY and XX/Sry ALCs, along with those of EGFP and mCherry, were examined using qRT-PCR. XY and XX/Sry testes were used as controls. Three biologically independent samples (n = 3) were used. The data were normalized to Actb and are presented as means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, using Student’s t-test (b). R software version 3.4.3 (https://www.r-project.org) was used to draw the plots in (b,c).