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.

Epididymal small non‐coding RNA studies: progress over the past decade

“…Then, one can use Percoll gradient centrifugation (Krapf et al, 2012;Nixon et al, 2015b) and/or somatic cell lysis buffer (Peng et al, 2012;Sharma et al, 2016;Chu et al, 2017) to obtain a purified sperm fraction. The Harris laboratory also exploits primary cultures of the caput, corpus, and cauda epididymis epithelial cells as an alternative (Leir et al, 2015;Browne et al, 2018). In the studies using small mammals such as mice, due to the limited amount of luminal fluid that can be extracted from each epididymis, the enrichment and purification processes are more susceptible to various contaminations including cell debris and other extracellular vesicles in the blood or lymphatic vessels, and therefore, extra prior steps such as perfusing the vasculature to minimize the blood contaminations are recommended.…”

“…In addition, the mixed epididymal epithelial cell population can be sorted by flow cytometry to enrich specific cell types, such as basal (Mandon et al, 2015) and clear cells (recent studies from Dr. Sylvie Breton's laboratory), or analyzed by single-cell sequencing techniques, as presented by Dr. Ann Harris's laboratory at the Epididymis 7. The Harris laboratory also exploits primary cultures of the caput, corpus, and cauda epididymis epithelial cells as an alternative (Leir et al, 2015;Browne et al, 2018). After hours of ex vivo manipulation, there remains a question whether and how the natural states of the sncRNAs in the epithelial cells are affected by the time of RNA extraction and library construction.…”

HNF1 regulates critical processes in the human epididymis epithelium

“…We identified hepatocyte nuclear factor 1 (HNF1) as a key epididymis epithelial transcription factor by bioinformatic analysis of genome-wide open chromatin in immature epididymis epithelial cells. The HNF1 motif was over-represented in these cells (2,7) and we show here that it is also over-represented in adult human caput epididymis epithelial (HEE) cells (8). HNF1α and HNF1β recognize the same DNA consensus sequence, which they occupy as homodimers or heterodimers.…”

“…None of the epididymides had extension of the testicular cancer, and all were placed in normal saline solution until further processing. Epithelial cells from the caput were isolated and established in culture as described previously (8). The majority of these cells express CFTR protein (8), consistent with principal cells (19).…”

“…The caput and cauda luminal environments are probably the most relevant for sperm maturation (17,18). Here we investigate the role of HNF1 in regulating gene expression and important biological processes in the caput epididymis epithelium using primary adult HEE cells (8). Following siRNA-mediated depletion of HNF1α and HNF1β we determined the contribution of these factors to the caput HEE cell transcriptome.…”