The expression and localization of V-ATPase and cytokeratin 5 during postnatal development of the pig epididymis

Abstract

Objective: We examined the localization and expression of H+ pumping vacuolar ATPase (V-ATPase) and cytokeratin 5 (KRT5) in the epididymis of pigs, expressed in clear and basal cells, respectively, during postnatal development.

Methods: Epididymides were obtained from pigs at 1, 7, 21, 60, 120, and 180 days of age; we observed the localization and expression patterns of V-ATPase and KRT5 in the different regions of these organs, namely, the caput, corpus, and cauda. The differentiation of epididymal epithelial cells was determined by immunofluorescence labeling using cell-type-specific markers and observed using confocal microscopy.

Results: At postnatal day 5 (PND5), the localization of clear cells commenced migration from the cauda toward the caput. Although at PND120, goblet-shaped clear cells were detected along the entire length of the epididymis, those labeled for V-ATPase had disappeared from the corpus to cauda and were maintained only in the caput epididymis in adult pigs. In contrast, whereas basal cells labeled for KRT5 were only present in the vas deferens at birth, they were detected in all regions of the epididymis at PND60. These cells were localized at the base of the epithelium; however, no basal cells characterized by luminally extending cell projections were observed in any of the adult epididymides examined.

Conclusion: The differentiation of clear and basal cells progressively initiates in a retrograde manner from the cauda to the caput epididymis. The cell-type-specific distribution and localization of the epithelial cells play important roles in establishing a unique luminal environment for sperm maturation and storage in the pig epididymis.

Keywords: Basal Cell; Clear Cell; Epithelium; Immunofluorescence; Luminal Environment.

Figure 1

Immunolocalization of B1-VATPase (red) and KRT5 (green) in the epididymis at postnatal day (PND) 180. (A) A pig epididymis section labeled with anti-B1-VATPase. Goblet-shaped clear cells were observed (arrows). (B) A pig epididymis section labeled with anti-KRT5. Basal cells were located at the base of the epithelium (arrowheads). (C and D) Negative controls showed no B1-VAPTase or KRT5 staining in the epididymis. Nuclei are labeled with DAPI (blue). V-ATPase, vacuolar ATPase; KRT5, cytokeratin 5; DAPI, 4′, 6-diamidino-2-phenylindole. Bars = 50 μm.

Figure 2

Progressive appearance of clear cells and basal cells from the VD to the caput of epididymides during the first three postnatal weeks. Cells in the epididymis were double-labeled for B1-VATPase (red) and KRT5 (green). (A–D) Epididymis at PND1. (E–H) Epididymis at PND7. (I–L) Epididymis at PND21. Yellow and white arrows indicate round- or goblet-shaped clear cells, respectively. Yellow and white arrowheads indicate projecting or dome-shaped basal cells, respectively. Nuclei are labeled with DAPI (blue). VD, vas deferens; V-ATPase, vacuolar ATPase; KRT5, cytokeratin 5; PND, postnatal day; DAPI, 4′, 6-diamidino-2-phenylindole. Bars = 50 μm.

Figure 3

Epididymis from PND60 to PND180. Cells in the epididymis were double-labeled B1-VATPase (red) and KRT5 (green). (A–D) Epididymis at PND60. (E–H) Epididymis at PND120. No labeled clear cells were observed in the VD at PND120. (I–L) Epididymis at PND180. Clear cells are only labeled in the caput epididymis. Yellow and white arrows indicate round- or goblet-shaped clear cells, respectively. Yellow and white arrowheads indicate projecting or dome-shaped basal cells, respectively. S, spermatozoa. Nuclei are labeled with DAPI (blue). PND, postnatal day; V-ATPase, vacuolar ATPase; KRT5, cytokeratin 5; VD, vas deferens; DAPI, 4′, 6-diamidino-2-phenylindole. Bars = 50 μm.

Figure 4

Quantitative changes in cell numbers during postnatal development of the pig epididymis. (A) Changes in the number of clear cells in the caput, corpus, and cauda. (B) Change in the volume of basal cells in the caput, corpus, and cauda. Cell numbers were calculated based on the number of clear and basal cells in the epithelium per square millimeter of the epididymal area. Results are expressed as means±standard error of the mean. Means with different letters differ (p<0.05).

Figure 5

Basal cell projections in the pig epididymis. The basal cells are labeled for KRT5 (red), and tight junctions are labeled for ZO-1 (green). (A–C) During the first 21 days of postnatal development, basal cell projections passed through the tight junctions, thereby coming into contact with the luminal contents (arrows). (D–E) The arrows indicate basal cells with short projections that do not reach the apical border of the epithelium. (F) The arrow indicates basal cells with no projections. The yellow arrowheads indicate tight junctions. Nuclei are labeled with DAPI (blue). KRT5, cytokeratin 5; ZO-1, zonula occludens-1; DAPI, 4′, 6-diamidino-2-phenylindole; PND, postnatal day. Bars = 20 μm.

Figure 6

Schematic diagram of epithelial cell differentiation in pig epididymis during postnatal development. (A) During early postnatal development, all epithelial cell types in the pig epididymis have a similar morphological appearance (round- or columnar-shaped). (B) During the pre-puberty stages of development, the different types of epithelial cell undergo morphological differentiation. Whereas principal cells maintain their columnar shape, basal cells occasionally develop narrow or triangular-shaped cytoplasmic cell bodies that can extend to the tight junctions. Moreover, the nuclei of these cells begin to migrate to the base of the epithelium. The morphology of clear cells changes from columnar- to goblet-shaped. (C) In the epididymis of adult pigs, the different types of epithelial cell are morphologically well differentiation. Principal cells have a columnar-shaped cell body, basal cells have a dome-shaped cell body, and clear cells have a goblet-shaped cell body.