Molecular Human Reproduction, Vol. 7, No. 10, 935-945,
October 2001
© 2001 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
Epididymal epithelium immortalized by simian virus 40 large T antigen: a model to study epididymal gene expression
1 IHF, Institute for Hormone and Fertility Research at the University, Hamburg, Germany and 2 Department of Reproductive Sciences and Medicine, Imperial College of Science, Technology, and Medicine, Hammersmith Hospital, London, UK
Abstract
Primary cultures of the differentiated, adult epididymal duct epithelium were immortalized by retroviral transduction with the simian virus (SV)40 large T antigen. The canine epididymis was chosen here as a model with high human relevance, representing a convenient and acceptable source of differentiated epididymal tissue and, compared to other animal models, expressing a relatively large number of gene products which are also expressed by the human epididymis. To determine whether the immortalized canine epididymal (IMCE) cells retained a phenotype comparable to the original tissue, epithelial cytokeratins, various epididymal transcription factors as well as mRNAs encoding abundant epididymal secretory proteins, were studied as molecular markers. All IMCE populations obtained after transduction were of epithelial origin. The nuclear androgen receptor (AR) and the polyoma enhancer activator (PEA3), as well as the epididymal mRNA encoding the canine counterparts of human HE1, HE4 and HE5/CD52 epididymal mRNA, were retained in all populations tested. The majority of tested clones were oestrogen receptor ER
-positive, but ERß-negative, while one ER-negative cell population was positive for ERß. The IMCE populations described thus represent useful permanent tools for studying gene expression of the epididymal duct epithelium, and for other types of experiments, examples including drug effects and toxicity on the epididymis.
epididymis/HE1/HE4/immortalization/transcription factors
Introduction
In most mammals, the epididymis has been shown to be indispensable for the acquisition of male fertility (Yanagimachi, 1994
). Although the epididymal duct epithelium is essential in this process, its precise function is still unknown. To elucidate its role and mode of regulation, various in-vivo and in-vitro models have been promoted. The different in-vitro approaches described include the culture of whole epididymal tubules (Orgebin-Crist et al., 1976; Klinefelter and Hamilton, 1984), epithelial cell aggregates (Byers et al., 1985; Moore et al., 1992; Raczek et al., 1994; Pera et al., 1996; Chen et al., 1998), and individual epithelial cells alone or in co-culture with fibroblasts or spermatozoa (Klinefelter et al., 1982; Finaz et al., 1992; Moore et al., 1992, 1998; Carballada and Saling, 1997). We have previously established primary cultures of the rodent and canine epididymal duct epithelium to study the regulation of epididymal mRNA expression (Pera et al., 1996; Kirchhoff et al., 2000). The cells showed characteristics of the native epididymal duct epithelium and could be maintained for sufficient periods of time for immediate experiments. They retained the nuclear androgen receptor (AR) and responded to androgen supply or withdrawal. Specifically, they provided a tool to demonstrate for the first time a direct temperature effect on the tissue-specific mRNA expression pattern of the epididymal epithelium and this was independent of temperature effects on the testis.
However, a major obstacle of these primary cultures is their relatively short duration and finite cell number due to a limited proliferative potential of the cells. Establishing a continuous supply of permanent epididymal epithelial cells without signs of senescence and suitable for molecular studies, therefore, remained a key issue. The objective of the present study was to generate immortalized epididymal epithelial cells by transduction with genes from a DNA tumour virus. The simian virus (SV)40 large T antigen is the most commonly used immortalizing oncogene suitable for almost any mammalian cell type (Ozer, 2000). Transduction of normal cells with this oncoprotein has been reported to inactivate various cell growth suppressors, e.g. p53 and Rb proteins (Manfredi and Prives, 1994; Jha et al., 1998; Yeager and Reddel, 1999).
Human epididymal tissue suitable and sufficient for cell culture and retroviral transfection is practically unavailable, but the canine epididymis offers a good alternative with high human relevance (Ivell et al., 1998). Not only does it represent a convenient and acceptable source of differentiated epididymal tissue suitable for various in-vitro studies (Ellerbrock et al., 1994; Pera et al., 1996), but also, compared to other animal models, it expresses a relatively large number of gene products which are also expressed by the human epididymis. Investigations into the mechanism whereby endocrine and paracrine signals affect epididymal functions depend on the degree to which the cultured cells retain the phenotype of their in-vivo counterparts. In this study, we have used epididymal mRNAs encoding secretory proteins and transcription factors as markers to determine whether the cells retain a phenotype comparable to the original tissue. The cloning of several canine homologues of human epididymal gene products has been described previously (Ellerbrock et al., 1994). Additional epididymal mRNAs have recently been cloned by the differential screening of a canine epididymal (CE) cDNA library (Beiglböck et al., 1998; Gebhardt et al., 1999) and used in turn for the cloning of a novel human epididymis-specific gene (Saalmann et al., 2001). In-situ transcript hybridization has shown that all mRNAs are transcribed by the epididymal duct epithelium (Pera et al., 1994; Beiglböck et al., 1998; Gebhardt et al., 1999; Saalmann et al., 2001). While several of these markers are lost already during the first days of primary culture prior to immortalization, we demonstrate here that CE1 and CE4, encoding the canine counterparts of human HE1 and HE4 epididymal secretory proteins (Kirchhoff et al., 1991, 1996), as well as various transcription factors implicated in the differentiated phenotype of the epididymis, are retained after transformation. These permanent cell lines thus represent a very useful tool for studying specific gene expression and regulation of the epididymal duct epithelium.
Materials and methods
Animal tissue
Canine epididymides and ovaries were obtained from local veterinary practices, where dogs were being castrated for behavioural disturbances. Epididymides from young, sexually mature and healthy outbred dogs (mongrels, 2–5 years old) were chosen for cell culture experiments. Tissues were removed at operation, cleaned of irrelevant tissue, transferred to Dulbecco's modified Eagle's medium (DMEM) medium (Gibco-BRL, Eggenstein, Germany) at room temperature, including 50 IU/ml penicillin (Sigma, Deisenhofen, Germany) and 50 µg/ml streptomycin (Sigma), and immediately processed for primary cell culture as described below. Control epididymides were snap-frozen in liquid nitrogen, either as entire organs or separated according to gross morphology into caput, corpus, cauda, and vas regions.
Primary cell culture
Whole individual epididymides were carefully freed from surrounding tissue, and the ducts were cut into small pieces and digested with 10 IU/ml Collagenase II (Sigma) for 2 h at 37°C. Small tissue fragments were collected by sedimentation and plated out in Matrigel (Collaborative Biomedical Products, Bedford, MA, USA) or bovine dermal collagen (Cellon; Strassen, Luxembourg) coated 6-well plates at a ratio of 500 mg fresh tissue weight per well. 24 h later, the DMEM plating medium and all unattached cells were aspirated, and the primary culture was maintained in a 1:4 mixture of Ham's F-12/DMEM without phenol red, charcoal-stripped fetal calf serum (1% v/v FCS; PAA Laboratories, Linz, Austria), penicillin (50 IU/ml), streptomycin (50 µg/ml), L-glutamine (2 mmol/l), 8-Br-cAMP (5 µmol/l), transferrin (10 µg/ml), epidermal growth factor (10 ng/ml), insulin (1 µg/ml), hydrocortisone (2 µg/ml), retinol (1 µg/ml), and selenium (17 ng/ml) (all ingredients from Sigma). Depending on the experiment, androgens were added as a mixture of 100 nmol/l testosterone (Sigma) plus 100 nmol/l dihydrotestosterone (DHT; Sigma). Primary cultures were routinely run at 33°C; during experiments on the effects of temperature a number of culture wells were additionally set up at 37°C. After 3 days, suitable primary cultures were selected and subjected to retroviral infection.
Retroviral infection
Cell cultures were allowed to grow to 80% confluence before application of viral load. In order to synchronize the cell cycle, the cultures were cooled to room temperature prior to infection. Two helper cell strains of mouse fibroblast cell line PA317 (Miller and Buttimore, 1986; a kind gift from Dr Nick Lemoin, Imperial Cancer Research Fund Oncology Unit, Hammersmith Hospital, London, UK), packaging retroviruses pBABE-v-myc or pZipSV40-6 respectively (Morgenstern and Land, 1990a,b), were raised in medium 199 (Sigma) plus 10% (v/v) FCS, 2 mmol/l L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin. At 50% confluency, the respective antibiotic was added (pBABE-v-myc, hygromycin; pZipSV40-6, neomycin). Retroviral infection was performed as described elsewhere (Brosens et al., 1996). Briefly, the PA317 culture medium was exchanged for the epididymal primary cell culture medium for 48 h without antibiotic, then this was taken off and applied to the primary cultures in the presence of 2 µg/ml polybrene (Sigma). Cells were exposed to the viral load for three days, applying fresh load approximately every 12 h. Only a few cells infected with pBABE-v-myc survived the viral load, and the remaining cells could not be passaged. Therefore the oncogene v-myc was not included in further experiments.
Selection of transformed cells
After the infection, cells were submitted to antibiotic selection employing G418 (Gibco-BRL) at a concentration of 1 mg/ml. From the surviving colonies, separable single colonies were scraped, dispersed, and allowed to grow in individual Petri dishes. Individual colonies were dispersed from cellular islands, and plated at a ratio of 1:0.3 in 96-well plates for clonal selection. From these plates, a first selection of 50 cell lines was chosen and grown in 6-well plates in culture medium containing 400 µg/ml G418. A second selection for cell lines resembling the phenotypes of the primary cell cultures was performed. Finally, six individual clones were chosen for detailed cellular characterization. These cells tested negative for mycoplasma by enzyme-linked immunosorbent assay (Boehringer–Mannheim GmbH, Mannheim, Germany). The transduced cells were routinely run at 37°C; during temperature experiments a number of culture wells were set up at 33°C in parallel. Cell numbers were estimated by counting using a bright light haematocytometer (Sigma).
Light microscopy and immunocytochemistry
Cells were grown on uncoated or Matrigel-coated Lab-Tek 4-chamber slides (Nunc, Roskilde, Denmark) for a minimum of 3 days. Primary cultures of the canine epididymis and 10 µm cryosections through canine epididymal tissue were used as controls. Cells and cryosections were fixed, permeabilized and either examined by light microscopy or processed for immunofluorescence and immunoperoxidase staining as described (Pera et al., 1996; Kirchhoff et al., 2000). For the detection of nuclear AR protein the rabbit polyclonal antibody PG21 (courtesy of Professor G.L.Greene, Chicago, USA) (Prins et al., 1991) was used at dilutions of 1:100 to 1:200 (~1–2 µg/ml). Cytokeratin intermediate filaments were stained using rabbit polyclonal antibody Z622 (Dako, Hamburg, Germany) at dilutions of 1:500 to 1:1000. Polyclonal EPV20 antibody from rabbit (courtesy of Dr Lotte Larsen, University of Aarhus, Denmark) was employed at a dilution of 1:1000 for detecting the CE1 protein. The actin monoclonal antibody (Sigma, A2547) was used at a dilution of 1:500. Normal rabbit IgG at comparable dilutions was included as an antibody control.
Protein preparation and Western blot analysis
Following a brief rinse in PBS, crude protein extracts were prepared by scraping epididymal cells ice-cold in RIPA lysis buffer (150 mmol/l NaCl, 1.0% v/v NP-40, 0.5% deoxycholate (DOC), 0.1% sodium dodecyl sulphate, 50 mmol/l Tris pH 8.0) supplemented with a proteinase inhibitor cocktail (Complete; Boehringer–Mannheim). Protein concentrations were measured spectrophotometrically using the BioRad DCC staining procedure (BioRad, München, Germany). Samples of ~15 µg protein per lane were separated on 12.5% polyacrylamide gels (Rittenhouse and Marcus, 1984), and transferred onto PVDF membranes (Amersham, Braunschweig, Germany) in a discontinuous buffer system using a semi-dry blotter (Phase, Lübeck, Germany). Immunodetection was carried out by blocking the membrane in 2.5% non-fat dry milk (Blotto; Boehringer–Mannheim, in TBS/0.005% Thimerosal) for 1 h, followed by an incubation with a polyclonal anti-large T antiserum (Santa Cruz; dilution 1:1000 in Blotto) at 4°C overnight. Antibody binding was recognized by a peroxidase-coupled secondary F'ab fragment antibody (Amersham). For chemiluminescence detection the CL-HRP substrate system (Pierce Chemical Company, Rockford, IL, USA) was used at a dilution of 1:10 + 0.3% BSA before exposure to X-ray-film (Fuji RX100, Tokyo, Japan).
RNA extraction, reverse transcription–polymerase chain reaction (RT–PCR) and Northern blot analysis
Medium was carefully aspirated from cell cultures, and cells were either lysed in 1 ml/well of the chaotropic solution TristarTM (AGS, Heidelberg, Germany) or according to the manufacturer's protocol for the RNAeasy system (Quiagen, Hilden, Germany). Total RNA yields were estimated photometrically, and equal amounts submitted to Northern blot analysis as previously described (Pera et al., 1996). Briefly, 5–10 µg of total RNA per lane, depending on the type of experiment, was separated by denaturing agarose gel electrophoresis and transferred to Hybond N+ nylon membranes (Amersham, Braunschweig, Germany). Equal loading was ascertained by ethidium bromide staining of gels prior to blotting. Digoxigenin (DIG)-labelled cDNA probes were prepared by PCR amplification of purified cDNA fragments following the instructions of the supplier (Boehringer, Mannheim, Germany). Complementary DNA was synthesized from 2–5 µg of total RNA extracts from tissue or cells according to the GeneScript protocol (Genecraft, Münster, Germany) in the presence of 0.5 mol/l betaine (Sigma) at 23°C for 10 min followed by a 30 min incubation at 42°C. Sequences of all primer pairs employed in RT–PCR are shown in Table I. PCR for CE transcripts was performed with the above-mentioned primers and 1 U Biotherme Taq polymerase (Genecraft) in a touchdown PCR programme descending from 61 to 51°C annealing temperature. PCR for the AR, PEA3 and ER were performed with a touchdown programme descending from 64 to 56°C. Amplicons were separated and visualized on 1% TAE agarose/ethidium bromide gels. The identity of inserts was confirmed by sequencing (MWG-Biotech, Ebersberg, Germany).
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Results
Morphological assessment of permanent canine epididymal cells
Primary cultures of the canine epididymal duct were established using a modification of the explant technique (Pera et al., 1996) in which the cells were grown on supports coated with extracellular matrix (Kirchhoff et al., 2000). Considering earlier observed age-dependent changes of canine epididymis-specific gene expression (Gebhardt et al., 1999), only epididymides from young, sexually mature animals (2–5 years of age) were employed in the primary cultures. Explants began to attach to the basement within 24 h, and cells began to spread after ~48 h. Phase contrast microscopy revealed that after 3 days, a monolayer of regular polygonal cells grew from the explants and exhibited a clear epithelioid morphology with no signs of fibroblast overgrowth. Firmly attached day 3 primary cultures were exposed to retroviral infection with the SV40-containing vector pZipSV40-6 (Morgenstern and Land, 1990a,b). Cells having integrated the vector were selected by their resistance to the neomycin analogue G418. Surviving cell populations were maintained in complete culture medium. In a first clonal selection, 50 transformed cell populations were established and named IMCE 1–50.
Expression by all neomycin-resistant cell populations of the transforming large T antigen was confirmed by Western blot analysis employing an anti-T antibody (data not shown). Phase contrast microscopy revealed that the transformed cells exhibited the morphological characteristics of cells of epithelial origin (Figure 1), comparable to the originating primary cultures. While in several permanent populations the individual cells looked cobblestone-like, forming a very dense monolayer, a more spindle-like cell shape was observed in other populations which grew less densely (Figure 1). Most of the permanent cell populations, however, reached considerably higher saturation cell densities than the primary cultures. The majority of transformed populations reached 45 passages after 15 weeks of continuous culture without signs of senescence or going through a marked crisis. In the presence of reconstituted basement membrane on the bottom of the culture wells, the spindle-shaped cell populations grew in a more reticulum-like manner. In the absence of basement membrane, they eventually formed multilayer, cyst- or tubule-like structures detaching from the anchorage basement (Figure 1).
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Six lines, IMCE 3, 7, 12, 20, 28 and 29, were chosen for closer examination by immunocytochemistry. Their epithelial origin was confirmed by positive indirect immunofluorescence for epithelial cytokeratins (Achtstätter et al., 1985
) surrounding their perinuclear region as a filament network (Figure 2). At the same time, these populations tested negative for smooth muscle -actin staining (not shown).
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Compared to the originating primary epididymal cells, the transformed cells showed increased growth rates. As growth properties of the primary cells were affected by temperature (Pera et al., 1996
), proliferation of the transformed cells was studied at the different culture temperatures of 33 and 37°C. IMCE clones 12 and 28 were chosen as an example of spindle-shaped and cobblestone-like cell types, respectively (cf. Figure 1
). At a temperature of 33°C, population doubling times ranged from 18 to 20 h for the cobblestone-like cells of IMCE 28 and from 28 to 30 h for the spindle-shaped cells of IMCE 12. At a culture temperature of 37°C, growth rates were considerably increased, with doubling times ranging from 8 to 10 h for IMCE 28 to ~16–18 h for IMCE 12.
Expression of the nuclear AR
Through the use of the PG21 polyclonal antibody (Prins et al., 1991), primary monolayers of canine epididymal cells had previously been shown to be immunopositive for the nuclear AR (Pera et al., 1996). Immunoperoxidase staining of the selected IMCE clones 3, 7, 12, 20, 28 and 29 showed the presence of the nuclear AR also in the transformed cells (Figure 3). As expected, the majority of immunoreactive AR protein was detected in the cell nuclei. Androgen supplementation or its withdrawal from the culture medium did not affect the intensity or distribution of immunoreactive AR in the cells (not shown). However, immunostaining patterns and intensities varied significantly between cell populations (Figure 3), with the cobblestone-shaped IMCE clone 28 showing the most pronounced nuclear staining pattern. Expression of the AR was confirmed by RT–PCR analysis with primers for the canine AR mRNA (Genbank accession no. AF197950) in IMCE lines 3, 7, 12, 20, 28 and 29. RNA preparations extracted from whole canine epididymal tissue (Figure 4) and from day 6 primary cell monolayers were included as controls (not shown). Again, no significant differences in AR mRNA levels were obvious in the RNA extracts from cells grown with or without androgens in their culture medium (not shown).
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RT–PCR analysis of other epididymal transcription factors
Oestrogen receptors are expressed widely in the male genital tract, and ER
has been shown to be important for normal epididymal function (Hess et al., 1997; Lee et al., 2000). Similarly, a member of the ETS family of transcription factors, the polyoma enhancer activator 3 (PEA3), has been implicated in the regulation of epididymis-specific gene expression (Drevet et al., 1998; Lan et al., 1999). To determine whether these transcription factors were expressed by the permanent populations, RT–PCR analyses were performed in IMCE lines 3, 7, 12, 20, 28 and 29 by employing oligonucleotide primer pairs specific to mRNA encoding the PEA3 transcription factor (Xin et al., 1992), and to mRNA of the oestrogen receptors ER (White et al., 1987) and ERß (Walther et al., 1999). As the canine homologous cDNA sequences of these transcription factors were not in the databases, the primer sequences chosen for PCR amplification were taken from evolutionarily highly conserved regions (Table I). cDNA prepared from total canine epididymides and rat caput fragments were included as positive controls during amplification of PEA3 cDNA, and cDNA from canine epididymides and ovaries were used during the analysis of ER and ERß cDNA. All IMCE lines were clearly positive for the PEA3 mRNA but appeared to differ in their status of ER and ERß mRNA (Figure 5). The identity of the amplified cDNA fragments was confirmed by sequence analysis. The canine 410 bp PEA3 cDNA fragment (AJ313194) was 86% identical to the corresponding mouse sequence (Xin et al., 1992); the 950 bp ER (AJ313195) and the 750 bp ERß (AJ313196) cDNA fragments showed 89 and 90% identity respectively.
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Expression of specific gene products in primary and transformed epididymal cells
Northern blot analyses were performed to examine whether the primary cell cultures paralleling those used for the immortalization procedure had retained their ability to produce any of the CE mRNA specific to the native, sexually mature canine epididymal duct in vivo. DIG-labelled cDNA hybridization probes for eight epididymis-specific mRNA of epithelial origin were included in the analysis, i.e. CE1, CE4, CE5/CD52 (Ellerbrock et al., 1994
), CE7/GPX5 (Beiglböck et al., 1998), CE8, CE9, CE10 (Gebhardt et al., 1999), and CE12 (Saalmann et al., 2001). Of these, CE1 and CE4 mRNA are known to be transcribed by the duct epithelium in nearly all parts of the canine epididymis (Pera et al., 1994), while the others show a pronounced and distinct pattern of regionalization along the length of the epididymal duct (Beiglböck et al., 1998; Gebhardt et al., 1999; Saalmann et al., 2001). CE1, CE4 and CE5 mRNA were retained in the short-term primary cell cultures (Figure 6). After long-term culture and cell passage, however, only CE1 and CE4 mRNA persisted at levels detectable by Northern blotting (not shown).
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The levels of the other CE mRNA were not stable in vitro, but declined rapidly with different half-lives. Decline of most of the CE mRNA levels was already initiated after 2 days of primary culture, as soon as the tissue explants had attached and cells began to spread on the substratum to form a monolayer. To possibly overcome this early loss of tissue-specific mRNA, retroviral infection of unattached epididymal tissue fragments was also attempted; however, this did not result in any G418-resistent cell populations. No obvious differences were observed between cell cultures grown in the presence or absence of androgens in the culture medium (Figure 6
), suggesting that androgens alone were not sufficient to maintain epididymis-specific mRNA expression in these cell cultures. Also, while the increased culture temperature of 37°C affected the half-lives of some of the CE mRNA (most pronounced with CE5 and CE10), it did not change the overall pattern of mRNA decline (data not shown).
All permanent cell populations included in the Northern blot analysis continued to express CE1 and CE4 mRNA, but no other CE mRNA at levels detectable by Northern blotting (Figure 7). Compared to the native tissue and to the early primary cultures (Figure 6), however, the levels of CE1 mRNA were reduced while their CE4 mRNA levels were markedly increased. The expression levels of both mRNA species appeared to vary slightly between individual IMCE lines (Figure 7). Employing the polyclonal EPV20 antiserum (Larsen et al., 1997) raised against the bovine counterpart of the HE1 protein, the presence of HE1-related antigen in monolayers of the IMCE clones was confirmed immunocytochemically (not shown). The more sensitive analysis technique of RT–PCR also revealed expression of the CE5/CD52 mRNA in all populations (Figure 8).
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Discussion
We have generated permanent cell populations of the differentiated, adult epididymal duct epithelium by retroviral infection of cells with the SV40 large T antigen. Whether the IMCE described here have a truly unlimited proliferative potential remains to be established. However, the magnitude of increase in their lifespan is already sufficiently great for most practical purposes. A common criticism of experiments carried out with isolated, cultured epithelial cells is that the results obtained do not reflect the native functions of these cells. Tissue-type epithelial organization, on the other hand, is probably only retained during organ culture with the attendant inability to separate epithelial from connective tissue responses. The immortalization process as initiated here by retroviral transduction, besides disrupting the organ, additionally involves the disruption and uncoupling of the endogenous cell cycle control by an oncogene, and thus represents both biologically and methodologically a major manipulation. A qualitative and cell-specific selection process therefore is mandatory in order to identify among the resistance gene-expressing cells the ones best retaining the properties of the origin tissue.
The transformed epididymal epithelial cells studied retained important morphological and functional characteristics of their tissue of origin. Previous immunohistochemical studies in the differentiated male excurrent ducts had revealed that the cytokeratins 8, 18 and 19 were predominant in epithelial cells lining the epididymal duct, and not found in connective tissue (Achtstätter et al., 1985). Indeed, the IMCE lines studied were all immunopositive for these epithelial cytoskeleton markers. These cells, if exhibiting functional similarity to the native epididymis, should also be able to transcribe epididymal epithelial mRNA. All IMCE lines studied, including clones 3, 7, 12, 20, 28 and 29, expressed the CE1 and CE4 mRNA, representing the canine homologues of HE1 (Kirchhoff et al., 1996) and HE4 (Kirchhoff et al., 1991).
HE1 is a highly conserved protein showing a unique structure with notable similarity to the major mite allergens (Ichikawa et al., 1998). It is abundantly expressed by the epididymal duct epithelium (Krull et al., 1993; Kirchhoff et al., 1996) and has been implicated in luminal cholesterol transfer/exchange (Okamura et al., 1999). Consistent with this observation, HE1 has recently been identified as the second gene of Niemann-Pick C disease involved in egress of cholesterol from lysosomes (Naureckiene et al., 2000). HE4 is a less-well-conserved secretory protein of the epididymal duct epithelium. It contains two tandem-arranged whey–acidic protein domains, showing similarity to the secretory leukocyte proteinase inhibitor (SLPI/ALP/HUSI-1) which has been demonstrated to have multiple functions relevant to innate host defence (Jin et al., 1997; Ashcroft et al., 2000). Like SLPI, HE4 is found in the secretions of various mucosal epithelia. Recently, it has been characterized as a tumour marker (Schummer et al., 1999). The mRNA to both HE1 and HE4 proteins have previously been shown to be abundantly expressed in nearly all parts of the canine epididymal duct (Pera et al., 1994). However, most of the other mRNA markers which show spatial restriction of their epithelial expression patterns in situ (Beiglböck et al., 1998; Gebhardt et al., 1999; Saalmann et al., 2001) were lost from the primary cultures and permanent cells with different half-lives. It seems that the region-specific expression pattern, which is characteristic of the native epididymal duct epithelium, is dependent on an intact milieu which is not retained in the in-vitro systems. From our results, however, it may be inferred that the permanent cells generated in this study reflected at least all functions of the originating primary cultures of the epididymal duct epithelium.
It is interesting that the CE5 mRNA was detected by RT–PCR in all IMCE lines, showing maximum levels in IMCE 29. (Differences in CE mRNA levels were only observed under non-saturation PCR conditions, whereas in Figure 8 saturating PCR conditions are shown.) This mRNA encodes the canine counterpart of the glycosylphosphatidylinositol (GPI)-anchored HE5/CD52 sperm membrane antigen (Kirchhoff, 1996; Schröter et al., 1999; Kirchhoff and Schröter, 2001). From the CE5 cDNA sequence (Ellerbrock et al., 1994) it may be deduced that the canine peptide is also GPI-anchored, despite its completely divergent mature peptide sequence. As GPI-anchored proteins are transported apically in most epithelia (Le Gall et al., 1995), expression of this gene may be related to a polarized epithelial cell phenotype. The formation of `domes' and cyst- or tubule-like structures in several IMCE lines is additional presumptive evidence that some polarized functions are being retained, and suggests that GPI-anchored CE5 may be transported apically in the cell lines expressing it.
As androgens are the classical hormones known to be pivotal in the regulation of many epididymal functions (Orgebin-Crist, 1996), it is of interest that all IMCE clones studied retained the AR mRNA and protein. Further studies will have to be performed to elucidate any direct androgen dependence of CE mRNA expression; however, the inability of all CE mRNA levels studied here to significantly respond to androgens, despite the presence of the nuclear AR, may reflect a partial loss of the differentiated phenotype. All IMCE clones studied were also positive for the PEA3 mRNA. PEA3 is a tissue-restricted transcription factor which has been shown to be highly expressed by the epididymis (Xin et al., 1992) with maximum levels observed in the proximal parts of the organ (Lan et al., 1997). Moreover, it has been implicated in the regulation of epididymis-specific gene expression (Drevet et al., 1998; Lan et al., 1999). It is of interest to note that PEA3-binding motifs have been found in the 5'-flanking regions of numerous epididymally expressed genes. Additionally, alternate expression of either ER or ERß mRNA was observed in the IMCE populations. While IMCE 7, 12, 20 and 29 were positive for ER but negative for ERß mRNA, IMCE 3 was positive for ERß and negative for ER mRNA. Thus, the use of IMCE lines during in-vitro promoter studies of their 5'-flanking regions could replace the co-transfection of heterologous cell systems with various transcription factors as well as the transfection of primary epididymal cell cultures.
From previous studies in rodents it would appear that in the adult epididymis most of the ER protein (immunoreactivity) is expressed in the efferent ductules, whereas the more distal parts of the organ contain considerably less of this transcription factor (Hess et al., 1997; Lee et al., 2000; Atanassova et al., 2001). In comparison, ERß is expressed in most, if not all, epididymal cell types at all stages studied (Hess et al., 1997). These findings in situ appear not to be consistent with our observation that more IMCE clones were positive for ER mRNA than for ERß mRNA. However, changes in the expression pattern of oestrogen receptors with age, epididymal region or hormone treatment are still under debate, and the description by different authors is conflicting (Lee et al., 1997; Hess et al., 2000; Atanassova et al., 2001). Nevertheless, it might well be that, concerning their ER expression, the IMCE clones exhibit a differentiation state more typical for the prepubertal epididymis (Atanassova et al., 2001).
The in-vivo transformation of the adult epididymis is a rare event, hence there are only very few reports on the occurrence of epididymal cancer in situ, with most valid cases representing adenocarcinomas (Jones et al., 1997). In-vitro transformation of the epididymal duct epithelium has been attempted rarely. The only previous report, describing the establishment of the two human male genital tract cell lines REP and RVP (Coleman and Harris, 1991), was based on undifferentiated, fetal cells of Wolffian duct origin. Thus, the IMCE lines described here, originating from the adult canine epididymal duct epithelium, represent the first cell lines generated from the adult epididymal duct epithelium. They should be useful for many types of study, including epididymal drug and toxicology testing as well as for in-vitro promoter studies of epididymis-specific genes, and could represent an enabling research tool for renewed efforts to optimize culture conditions for polarized growth of epididymal epithelial cells.
Acknowledgements
We thank Ms Beate Harms and Ms Annemarie Samalecos for expert technical assistance. Dr Ilka Kascheike helped with the primary epididymal cultures in the beginning of the study. Professor G.L.Greene, Chicago, USA, generously provided the PG21 antibody; Dr Lotte Larsen, Aarhus, Denmark, the EPV-20 antibody; Dr Nick Lemoin, Hammersmith Hospital, London, UK, provided the PA317 helper cell lines. We are also very grateful to Professor Freimut Leidenberger for his continued interest and support. The study was supported by a DFG grant Ki317/5-2 and by an Ernst Schering Research Foundation grant given to R.T.
Notes
3 To whom correspondence should be addressed at: IHF Institut für Hormon- und Fortpflanzungsforschung, Grandweg 64, D-22529 Hamburg. E-mail: kirchhoff{at}ihf.de 
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Submitted on March 29, 2001; accepted on July 6, 2001.
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