Fig. 4. A center-to-pole pattern of glycogen accumulation along anteroposterior (AP) axis of developing XY gonads and close association between glycogen-rich cells and germ cells during early phases of testis differentiation. (A) Sagittal sections of XY gonads isolated at 14 ts (upper) and 15 ts (lower) stages. PAS staining. Regions I-V are equally divided along the AP axis of the gonad. PAS-positive cells at 14 and 15 ts are located in regions II and III, respectively. Interestingly, they are frequently found in an area near germ cells even at 14 and 15 ts stages (right panel). Right panels show higher magnification images, indicated by the boxed area in the corresponding left panel. Asterisk, germ cell; ms, mesonephros. Bar, 50 µm. (B) Transmission electron micrographs showing a direct association between glycogen-positive cells and germ cells in XY gonad at 15 ts. Right panel shows a higher magnification image, indicated by the broken rectangle in left panel. Arrows indicate glycogen granules. ce, coelomic epithelial cells; G, germ cells; GR, glycogen-rich cells. Bar, 5 µm. (C) All consecutive sagittal sections were stained with PAS reaction, and then the total number of positive cells in each gonad was measured separately in regions I-V, which were equally divided along the AP axis of the gonad (broken lines in A). The vertical axis represents the PAS-positive cell number per gonad, whereas the horizontal axis represents regions I-V of the gonads. In each graph, bars of the same color show the cell number in each region of the same gonad (14 ts, five gonads; 15 and 16 ts, each four gonads). PAS-positive cells are positioned predominantly in the middle regions (regions II to IV) at 14 ts. This center-restricted distribution clearly expands into the anterior (region I) and posterior (region V) edges from 15 to 16 ts.