Characterization of primary cultures of adult human epididymis epithelial cells

Abstract

Objective: To establish cultures of epithelial cells from all regions of the human epididymis to provide reagents for molecular approaches to functional studies of this epithelium.

Design: Experimental laboratory study.

Setting: University research institute.

Patient(s): Epididymis from seven patients undergoing orchiectomy for suspected testicular cancer without epididymal involvement.

Intervention(s): Human epididymis epithelial cells harvested from adult epididymis tissue.

Main outcome measure(s): Establishment of a robust culture protocol for adult human epididymal epithelial cells.

Result(s): Cultures of caput, corpus, and cauda epithelial cells were established from epididymis tissue of seven donors. Cells were passaged up to eight times and maintained differentiation markers. They were also cryopreserved and recovered successfully. Androgen receptor, clusterin, and cysteine-rich secretory protein 1 were expressed in cultured cells, as shown by means of immunofluorescence, Western blot, and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The distribution of other epididymis markers was also shown by means of qRT-PCR. Cultures developed transepithelial resistance (TER), which was androgen responsive in the caput but androgen insensitive in the corpus and cauda, where unstimulated TER values were much higher.

Conclusion(s): The results demonstrate a robust in vitro culture system for differentiated epithelial cell types in the caput, corpus, and cauda of the human epididymis. These cells will be a valuable resource for molecular analysis of epididymis epithelial function, which has a pivotal role in male fertility.

Keywords: Epididymis epithelial cell culture: caput; cauda; corpus.

FIGURE 1

Phase-contrast imaging of adult human caput, corpus, and cauda primary cells in culture (scale bar = 100 μm). (Top) One day after plating on collagen-coated plastic substrate. (Middle) By 3 days after plating, the cells flattened and started to grow as a monolayer. (Bottom) Cells at confluence. Leir. Human epididymis epithelial cell cultures. Fertil Steril 2014.

FIGURE 2

Regional gene and protein expression of epididymal markers in primary human epididymis epithelial cells from caput, corpus, and cauda. (A) Gene expression: total RNA extracted from cultures at passage 3 or 4 from caput, corpus, and cauda. Quantitative reverse-transcription polymerase chain reaction was performed with the use of Sybr Green with primers for the genes shown. Data are combined from cultures from two independent sets of tissue samples and are normalized to β2-microglobulin. (B) Protein expression: cell lysates separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blots probed with antibodies specific for androgen receptor (AR), clusterin, and cysteine-rich secretory protein 1 (CRISP1). β-Tubulin provided the loading control. Leir. Human epididymis epithelial cell cultures. Fertil Steril 2014.

FIGURE 3

Cysteine-rich secretory protein 1 (CRISP1), clusterin, cytokeratin 8 (scale bar = 20 μm), and cystic fibrosis transmembrane conductance regulator (CFTR; scale bar = 5 μm) expressed in primary human epididymis epithelial cells. Immunofluorescence with the use of specific antibodies as shown in caput, corpus, and cauda cells. Alexa Fluor 488–conjugated antirabbit IgG (green) or Alexa Fluor 549–conjugated antimouse IgG (red) was used as the secondary antibody. Cells were counterstained with 6-diamino-2-phenylindole (DAPI). CFTR in polarized caput cells is shown by means of confocal imaging. Leir. Human epididymis epithelial cell cultures. Fertil Steril 2014.

FIGURE 4

Effects of testosterone and R1881 on the transepithelial resistance (TER) of human epididymal epithelial cultures. (A) TER measurements in caput, corpus, and cauda cells cultured on membrane filter supports. (B) Immunofluorescence of androgen receptor (AR) in primary caput HEE cells treated with R1881 for 16 hours. R1881 treatment induced AR relocation from the cytoplasm to the nucleus. 6-Diamino-2-phenylindole (DAPI) was used for nuclear counterstaining (scale bar = 50 μm). (C) Effects of testosterone and R1881 on the maintenance of the tight junction permeability barrier of the caput. The resistance was multiplied by the effective growth area of the filter (~0.31 cm²) to yield the area resistance (ohm-cm²). The net value of the TER was computed by subtracting the background. n = 3; representative experiment shown. Leir. Human epididymis epithelial cell cultures. Fertil Steril 2014.