At PND1, B1-VATPase-labeled CCs were observed from the proximal vas deferens to the cauda and corpus regions of the epididymis (
Figure 2B–2D), although they were not detected in the caput (
Figure 2A). Within the vas deferens, we observed numerous CCs characterized by a goblet-shaped morphology (
Figure 2D, white arrows). In the epididymis, however, CCs with a round shape or goblet-like appearance were only occasionally detected in the corpus and cauda, respectively (
Figure 2B, yellow arrow;
Figure 2C, white arrows). At PND7 and 21, goblet-shaped CCs were detected in the cauda and vas deferens (
Figure 2G, 2H, 2K, and 2L, white arrows), whereas a small number of CCs with a round morphology were detected in the caput and corpus (
Figure 2E, 2F, 2I, and 2J, yellow arrows). At PND60, we observed that CCs continued to appear progressively in an ascending pattern from the distal region (cauda) to the proximal region (caput) of the epididymis (
Figure 3A–3D). However, the morphology of the CCs tended to vary depending on the regional location. Round- and goblet-shaped CCs were mainly present in the caput (
Figure 3A, yellow arrows) and cauda (
Figure 3C, white arrows), respectively. Within the corpus, we observed both round- and goblet-shaped CCs (
Figure 3B). These results indicate that the differentiation of CCs commences from the distal region (cauda) and proceeds to the proximal region (caput) of the epididymis. At PND120, goblet-shaped CCs were observed in all regions of the epididymis (
Figure 3E–3G, white arrows). However, we noted a marked decrease in the number of CCs in the cauda (
Figures 3G,
4A) and found that they were no longer detectable in the vas deferens (
Figure 3H). At PND180, CCs were observed only in the caput (
Figures 3A,
4A), all of which were goblet-shaped, and were absent from the vas deferens to the corpus (
Figure 3J–3L). Again, these observations indicate that the differentiation of CCs is initiated in the distal region of the epididymis and subsequently proceeds to the proximal region. No labeling was detected when the B1-VATPase primary antibody was omitted from tissue preparations (
Supplementary Figure S1A). The changes in the numbers of CCs were examined according to region and age (
Figure 4A). In the caput, the number of CCs peaked at PND120 and then significantly decreased at PND180 (
Figure 4A). In the corpus and the cauda, quantification analysis (
Figure 4A) showed that the number of CCs peaked at PND60 and then considerably decreased at PND120. We did not observed CCs at PND180 from both the corpus and the cauda regions of the epididymis (
Figure 4A).