Our understanding of epididymal development and cellular differentiation has relied extensively on early morphological studies characterizing changes in the structure of undifferentiated short columnar cells, and their differentiation into basal cells and tall columnar/narrow cells which subsequently develop into principal, clear, narrow and apical cells. The origin of epididymal dendritic cells remains to be clearly established, but these may represent cells initially identified by Cooper et al (Holschbach and Cooper, 2002) as blood-derived basal cells. Recent findings showed that epididymal basal cells share common properties with adult stem cells and can differentiate in vitro (Mandon, Hermo and Cyr, 2015a), in vivo using subcutaneous basal cell implants (Mou, Vinarsky, Tata, Brazauskas, Choi, Crooke, Zhang, Solomon, Turner, Bihler, Harrington, Lapey, Channick, Keyes, Freund, Artandi, Mense, Rowe, Engelhardt, Hsu and Rajagopal, 2016), and in basal cell-derived organoids under 3D cell culture conditions (Pinel, 2020). These recent observations have resulted in a need to re-evaluate cellular differentiation in the epididymis.
Epididymides of 7-day old rats are comprised of undifferentiated short columnar cells. Transcripts expressed at day 7 are heavily enriched with genes implicated in the cell cycle and stem cell signalling, supporting the previously reported morphological observations that the epithelium is largely undifferentiated at this age. Our microarray data demonstrate that numerous genes were highly expressed almost exclusively at day 7, including some of the gap junction proteins, cadherins (eg. CDH3, CDH5, and CDH6), claudin6 (CLDN6), a tight junction protein, and multiple proteins implicated in WNT signalling (WNT9b, Wnt6, LGR5, FZD2 and FZD10). CDH2 and CDH3 expression in undifferentiated columnar cells is interesting. These genes have been shown to be expressed in stem cells of the mammary gland, osteoblasts, chondrocytes, pro-myocytes and others (Alimperti and Andreadis, 2015, Patil, 2021, Ranjan, et al., 2021). Curiously, following the differentiation of basal cells, these genes become localized specifically to basal cells. LGR5 has been reported as a marker of adult stem cells in multiple tissues, including the intestine, bronchioles, mammary gland, and prostate (Barker and Clevers, 2010, de Visser, et al., 2012, Leung, Tan and Barker, 2018, Schuijers and Clevers, 2012, Seishima, et al., 2019).
Cellular differentiation of the epididymis is accompanied by dramatic changes in gene expression as the epithelium proliferates and differentiates. The largest changes in gene expression occurred between days 7 and 18, during which 1452 genes were differentially expressed, with 787 genes being down regulated and 665 genes being up-regulated. Downregulated genes at day 18 vs day 7 represented the major group of genes of the microarray analyses, showing the unique signature of the columnar cells.
Crisp1 (cysteine-rich secretory protein 1) and serpina16 (serine peptidase inhibitor, Clade A member 16) genes, also known as rat HongrES1(Ni, et al., 2009, Zhou, et al., 2008), showed the highest increase in mRNA levels between days 7 and 18 with over 95-fold increase, while Bin2a (beta-galactosidase-like protein), the third highest, showed a 63-fold increase (Supplemental Table III). All three genes have been shown to be androgen-responsive in the epididymis (Brooks, et al., 1986, Zhen, et al., 2009, Zhou, Zheng, Shi, Zhang, Zhen, Chen and Zhang, 2008). Other androgen-regulated genes such as Lcn5 (lipocalin 5), Adam7 (a disintegrin and metallopeptidase domain 7), DEFB1 (defensin beta 1), AQP9, Spink13 (serine peptidase inhibitor, Kazal type 13), and several DEFB (Hu, et al., 2014, Ma, et al., 2013) were also highly upregulated from 7 to 18 days (Lareyre, et al., 2000, Ma, Yu, Ni, Hu, Ma, Chu, Liu and Zhang, 2013, Oh, et al., 2005, Oliveira, et al., 2005, Palladino, et al., 2004), suggesting that early development is strongly influenced by androgens, even though androgen levels have been reported as being low at this age (Scheer and Robaire, 1980). Furthermore, while transcripts for the androgen receptor increase only slightly by day 18, there is a 10-fold increase in type 2 4-ene-5-alpha-reductase (SRD5a) (Viger and Robaire, 1995, Viger and Robaire, 1996). While expression levels of many of these genes further increase by day 28, the results show that androgen regulation in the epididymis begins at these early stages of development, prior to the presence of morphologically distinct principal cells (Sun and Flickinger, 1979).
Of the 787 down regulated genes in the 7 to 18-day period, 41 were downregulated by a factor of 5-fold of more. The transcription factor FOS (FBJ osteosarcoma oncogene), which has been shown to be androgen-repressed (Shankar, et al., 2016), displayed the highest degree of downregulation, 14.5-fold. In contrast to the upregulated genes associated with androgen regulation in the epididymis, there was no obvious pattern of regulation with the most highly downregulated genes.
Interestingly, several imprinted genes were downregulated at 18 days: DLK1 (delta like non-canonical Notch ligand 1) with a fold change of -7.75, the GNAS complex locus with a fold change of -6.79, and IGF2 (insulin-like growth factor 2) with a fold change of -6.26. DLK1 regulates cell differentiation and developmental processes through Notch and other signalling pathways (Traustadottir, et al., 2019). Igf-2 is almost exclusively expressed in the embryo of rodents (Chao and D'Amore, 2008). AGTR2 (angiotensin II receptor, type 2), which is a basal cell marker, was also decreased (fold change of -4.8, (Shum, et al., 2008)), as was Osr1 (odd-skipped related transcription factor 1; fold change of -4.35), a transcription factor implicated in normal development and organogenesis of the heart and kidney (Wang, et al., 2005). Numerous V-ATPases were found to be upregulated at day 18 (Fig. 3A).
During the period of cell differentiation, days 18 and 28, our results indicated 344 upregulated and 327 downregulated genes. Of the most highly expressed genes, epididymis-specific Gpx5 (glutathione peroxidase 5), an androgen- and testicular factors-regulated gene (Drevet, et al., 1998), showed the highest increase, with a fold change of 62.61. Rat Gpx5 transcripts were previously detected at day 32 by RT-PCR, before the appearance of the protein in the epididymis at day 44, just prior to puberty and the entry of sperm in the epididymis (Williams, et al., 1998). Other sperm-binding molecules and androgen-regulated genes also showed increased transcripts levels, including multiple defensins, Crisp4 and Pate (prostate and testis expressed) proteins (Hu, Zou, Yao, Ma, Zhu, Li, Chen and Sun, 2014, Jalkanen, et al., 2005, Turunen, et al., 2011). The Gammaaminobutyric acid (GABA-A) receptor pi (GABRP) was decreased by 9.48-fold. This receptor subunit has been shown to be implicated in airway epithelial progenitor cell differentiation (Wang, et al., 2021). WNT9b (wingless-type MMTV integration site family, member 9B), CLDN6 (claudin 6) and CFTR (cystic fibrosis transmembrane conductance regulator) were also downregulated from 18 to 28 days, with a fold change of -6.25, -4.22, and -3.19, respectively.
There were 406 upregulated and 154 downregulated genes between days 60 and 28. Many of these included defensins (Ribeiro, Silva, Hinton and Avellar, 2016), lipocalins (Fouchecourt, Lareyre, Chaurand, DaGue, Suzuki, Ong, Olson, Matusik, Caprioli and Orgebin-Crist, 2003) and a transcript encoding a protein of the specialized mitochondria present in the midpiece of the sperm flagella, SMCP (sperm mitochondria-associated cysteine-rich protein (Kleene, Wang, Cutler, Hall and Shih, 1994). SMCP transcripts are present in round spermatids, where they are translationally repressed and are transcribed in elongated spermatids (Kleene, 1989). Légaré et al. (Legare, et al., 2017) detected the SMCP transcripts in the bovine caput epididymides.
Pathway associations using Metascape analyses indicated that in epididymides of 7 to 18-day old rats, there was a significant enrichment of regulated genes implicated in stem cells, estrogen, thyroid hormone and kidney development. This is consistent with previous observations that have reported the differentiation of small epididymal columnar cells into other cell types, as well as implicating a role for estrogens and thyroid hormones in epididymal development (Anbalagan, et al., 2010, De Paul, et al., 2008, Hess, Sharpe and Hinton, 2021, St-Pierre, et al., 2003, Sun and Flickinger, 1979). Similarities between epididymal and renal development may be related to the common embryonic mesonephric origin of both tissues. The influence of estrogens on the epididymis appeared to extend throughout all phases of development and cell differentiation (Hess, 2000, Hess, Sharpe and Hinton, 2021). Unlike estrogens, androgen-dependent genes displayed significant changes between days 28 and 18 and 60 and 28. However, some androgen-dependent epididymal genes showed considerably increased gene expression between days 28 and 18, and may represent highly sensitive transcriptional activation of certain genes to low levels of androgens. Surprisingly, there were no significant associations in cell junctions, epidermal growth factor or immune function genes. While many of these genes showed differential expression, it is possible that only a few key specific genes are altered during development, or that these occur in a segment-specific manner and were not detected with the current experimental protocol using the entire epididymis.
Immune cells of the epididymis, such as dendritic cells and macrophages, can produce inflammatory cytokines implicated in the immune response. Transcript levels for the cytokines IL1 alpha and beta, IL6, IL10, IL12 and IL20, as well as for interferon gamma and tumor necrosis factor, were expressed at low levels throughout development. Expression of the tryptophan-depleting enzymes (indoleamine 2,3-dioxygenases (Ido1 and Ido2) increased during epididymal development. These enzymes are recognized to be implicated in the regulation of the equilibrium between the immune response and self-tolerance (Jrad-Lamine, et al., 2011, Mehraj, et al., 2020), which is an important mechanism for sperm maturation. Transforming growth factor-beta ligands 1, 2 and 3 were expressed at similar levels at day 7 in the epididymis. Transforming growth factor beta1 (TGFb1) mRNAs showed a constant level of expression throughout development, while TGFb2 and 3 mRNA levels decreased slightly. The TGFb pathway has been shown to be implicated in the immunotolerance to sperm in mice (Pierucci-Alves, et al., 2018).
Innate immune receptors to pathogens, Toll-like receptors (TLRs) 1 to 11 are expressed in the epididymis (Liman, et al., 2019, Munipalli, et al., 2019, Rodrigues, et al., 2008). Microarray data indicated that TLR3 and 5 were the most highly expressed in the epididymis. TLR3 has the highest level of expression throughout the development, and TLR5 increased with age. TLR8 and 9 were not probed by the microarray. Nuclear factor kappa B subunits 1 and 2 (NFkB1 and NFkB2) were detected at all time points of epididymal development used in this study. The alpha inhibitor (NFkBia) was also highly expressed at days 7 and 18 and decreased at days 28 and 60 (Gregory and Cyr, 2014).
The widespread immunostaining for V-ATPase observed in undifferentiated organoids derived from CD49f+ epididymal cells from day 7 rats suggests that as the epididymal epithelium develops postnatally, many cells destined for further differentiation express this protein. Whether or not this is a transient type of previously-undescribed cell, or rather a stage of basal-type cells, is unknown. Numerous V-ATPases were also upregulated between day 7 and 18. There are reports in the literature regarding the expression of V-ATPase (various isoforms) as a function of differentiation in multiple tissues and/or cell types. Wissel et al (Wissel, et al., 2018) have reported a V-ATPase-NOTCH regulatory loop in Drosophila neural stem cells or neuroblasts. They and others suggest that V-ATPase and NOTCH act in multiple stem cell lineages during nervous system and adult gut development. Although various subunits or isoforms of V-ATPase family members are implicated in many cellular processes (McGuire, et al., 2017, Sun-Wada and Wada, 2015), the authors of the Drosophila studies suggest that the V-ATPase complex is required for self-renewal-of the neuroblast stem cells. Furthermore, they report that it is among the first and most-significantly down-regulated complex in differentiating daughter cells, and that it is necessary for maintaining NOTCH pathway activity.
V-ATPase (multiple isoforms) is associated with luminal acidification, which is critical in the epididymis and other tissues (Breton and Brown, 2013, Shum, et al., 2011). This luminal acidification is necessary for preserving spermatozoa in a quiescent state. However, multiple isoforms of V-ATPase in the epididymis have been reported in the literature (Pietrement, et al., 2006), which prompts the question of whether or not these multiple isoforms have different functions. V-ATPase subunits can interact with some enzymes that are part of the glycolytic pathway in mammalian cells (Breton and Brown, 2007). Intriguingly, another group has examined the roles of glucose metabolism, fatty acid oxidation, and reactive oxygen species (ROS) in the equilibrium between quiescence and self-renewal of adult stem cells (Mohammad, et al., 2019). The authors point out that low ROS is a hallmark of adult stem cells. This would align with a role for V-ATPase in maintaining pH and protecting cells from ROS damage, as well as promoting self-renewal of stem cells. Nonetheless, the fact that organoids derived from basal cells of adult rats also expressed V-ATPase to a significant degree (Pinel, 2020), as well as organoids cultured in either epididymal basal cell medium or the commercial kidney medium, further supports the notion that the expression of V-ATPase is, at the very least, an indicator of self-renewal of at least a subset of stem cells, and perhaps also suggests a transient phase of cellular differentiation.
In conclusion, the development of the epididymal epithelium and subsequent cellular differentiation into multiple specialized cell types is a complex, highly-coordinated process which relies on a variety of cues from the physiological environment as well as from numerous signalling pathways. We have identified multiple key signalling pathways involved in the various stages of epididymal epithelial development and differentiation. Information from our microarray and PCR analyses highlight the genes which play major roles at specific time points during development. Furthermore, we have developed a new in vitro model of epididymal organoids, derived from undifferentiated Cd49f-positive columnar cells. Finally, we have provided evidence of the stem cell-like properties of these cells, and have provided methods for both proliferation as well as differentiation of these columnar cells into other cell types. We believe that these data significantly advance our understanding of epididymal epithelial development and differentiation.