Characterization of a Novel Rat Epididymal Cell Line to Study Epididymal Function
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《内分泌学杂志》
Institut National de la Recherche Scientifique-Institut Armand-Frappier (J.D., N.S., D.G.C.), Université du Québec, Pointe-Claire, Québec, Canada H9R 1G6
Ontogeny-Reproduction Research Unit (R.S.V.), Centre Hospitalier Universitaire Laval Research Centre and Centre de Recherche en Biologie de la Reproduction, Department of Obstetrics and Gynecology, Université Laval, Sainte-Foy, Québec, Canada G1K 7P4
Department of Anatomy and Cell Biology (L.H., D.G.C.), McGill University, Montreal, Québec, Canada H3A 2B2
Abstract
The epididymis is an androgen-dependent organ that allows spermatozoa to become fully functional as they pass through this tissue. The specialized functions of the epididymis are mediated by interactions between epididymal epithelial cells and between epididymal cells and spermatozoa. Although the critical role of the epididymis in sperm maturation is well established, the mechanisms regulating cell-cell interactions remain poorly understood because of the lack of appropriate cell line models. We now report the characterization of a novel rat caput epididymal cell line (RCE) that was immortalized by transfecting primary cultures of rat epididymal cells with the simian virus 40 large T antigen. At the electron microscope level, the cell line was composed of epithelial principal cells with characteristics of in vivo cells; principal cells had well-developed Golgi apparatus, abundant endoplasmic reticulum cisternae, and few endosomes. RCE cells expressed the mRNAs coding for the androgen receptor, estrogen receptor , and 4-ene-steroid-5--reductase types 1 and 2 as well as epididymal-specific markers Crisp-1 and epididymal retinoic acid binding protein. Epididymal retinoic acid binding protein expression was significantly induced with dihydrotestosterone, although this effect was not blocked by flutamide, suggesting that RCE cells are not androgen responsive. Neighboring cells formed tight and gap junctions characteristic of epididymal cells in vivo and expressed tight (occludin and claudin-1, -3, and -4) and gap junctional proteins (connexin-26, -30.3, -32, and -43). The RCE cell line displays many characteristics of epithelial principal cells, thus providing a model for studying epididymal cell functions.
Introduction
IN MAMMALS, TESTICULAR spermatozoa do not have the ability to fertilize or swim. The acquisition of these properties (sperm maturation) occurs as spermatozoa transit through the epididymis (1, 2, 3). The epididymis is an androgen-dependent organ whose structure and functions rely on hormones and other regulatory factors mainly secreted by the testes (4, 5). Morphologically, the epididymis can be subdivided into four distinct anatomical regions: the initial segment, the head (caput), the body (corpus), and the tail (cauda) (6). The complement of cell types that compose the epididymal epithelium varies according to the region being studied (1). The four major epididymal epithelial cell types have been referred to as principal, narrow, basal, and clear cells based on structural and functional parameters. Principal cells are by far the most abundant and are active in the transport and secretion of ions and small organic molecules, protein synthesis and secretion, and the absorption of fluid (1). The secretory products of these cells appear to be the major players in modifying spermatozoa and in the attainment of their maturational characteristics. The most active regulator of principal cell function is dihydrotestosterone (DHT), which is synthesized via the 5-reduction of testosterone occurring mainly in the initial segment (4, 5).
Cellular interactions in the epididymis are critical for the formation of a specialized blood-epididymal barrier between epithelial cells, and this involves mainly interactions between principal cells, the major epithelial cell type. This barrier creates a unique microenvironment in the epididymal lumen that is crucial for sperm maturation (7, 8, 9). In this way, ions, solutes, proteins, lipids, and androgen concentrations are markedly different from the blood plasma (8). Communication between epithelial cells is also believed to be important in coordinating epididymal function (7, 8, 9). In the epididymis, cellular interactions are under the influence of several endocrine and paracrine factors originating from the testis that ultimately regulate epididymal gene expression (10, 11). The complexity of these interactions is a strong indication that they are crucial for epididymal sperm maturation. Thus, demonstrating how direct cellular interactions in the epididymis are regulated and their significance to epididymal physiology remains a major but crucial challenge for our understanding of epididymal function and its role in sperm maturation.
Several in vivo and in vitro models have been employed to study epididymal functions. The majority of in vitro models have relied on the use of organ cultures, tubule cultures, and primary cell cultures (12, 13, 14, 15, 16, 17, 18). As a whole, these approaches have generated information primarily on the expression and regulation of epididymal secretory proteins. Because primary cultures of epididymal epithelial cells have a short half-life in vitro, and because these cells divide very slowly and can dedifferentiate, this approach has important limitations for the study of epididymal functions (19). Given the complexity of cell-cell interactions and varied epididymal functions within the epididymis, an immortalized epididymal cell line would therefore provide essential and invaluable tools for the study of these interactions and their contribution to epididymal functions.
A recent study by Telgmann et al. (20) reported the immortalization of canine epididymal cells by transfecting primary epididymal cultures with plasmids expressing either the simian virus 40 large T antigen (SV40 LTAg) or c-myc. The canine cell lines expressed the androgen receptor (AR), estrogen receptor- (ER), and the polyoma enhancer activator (PEA3) as well as the canine counterparts of the human HE-1, HE-4, and HE-5/CD52 genes (20). Cell lines have also recently been derived from the mouse epididymis. The first of these was generated from mice harboring a temperature-sensitive SV40 LTAg transgene (21). Much like the canine cells, these mouse epididymal cells also expressed the AR and some epididymal-specific proteins such as phosphatidylethanolamine-like binding protein, epididymal retinoic acid binding protein (E-RABP), and epididymal protein of 17 kDa (EP17) (21). Sipila et al. (22) also reported several mouse epididymal cell lines that were derived from transgenic animals expressing an SV40 LTAg transgene under the control of the glutathione peroxidase-5 promoter. Although many of these cells expressed AR, ER, and some epididymal proteins, few of the cell lines expressed more than two epididymal-specific proteins. The ultrastructure of these cells and how they compared with the normal mouse epididymis also remained unknown.
In the present study, we report the characterization of a novel rat caput epididymal cell line (named RCE) that was immortalized by transfecting primary cultures of rat epididymal cells with the SV40 LTAg. Importantly, the RCE cell line consists predominantly of principal cells that retain many characteristics, both structurally and functionally, of principal cells in vivo, making them ideally suited for studying epididymal cell function in relation to sperm maturation.
Materials and Methods
Primary epididymal cell cultures
Sprague-Dawley rats (40 d old; 151–175 g) were purchased from Charles River Canada Inc. (St. Constant, Québec, Canada). Rats were maintained under a constant photoperiod of 12 h light and 12 h dark and received food and water ad libitum. All animal protocols used in this study were approved by the University Animal Care Committee.
Rats were euthanized by CO2 asphyxiation, and epididymides were dissected from the animals under aseptic conditions and placed in DMEM/HAM’s F12 culture media containing penicillin (50 U/ml) and streptomycin (50 μg/ml) (Sigma-Aldrich, Mississauga, Ontario, Canada). The tissue was cleared of fatty materials, and the proximal region of the epididymis was retained (initial segment and caput epididymidis). Cells from these segments were isolated according to the methods of Kirchhoff et al. (23). Briefly, epididymides from four rats were finely cut with scissors. Tissue fragments (2–3 mm) were placed in media containing the antibiotics and collagenase (2 mg/ml; Life Technologies, Inc., Burlington, Ontario, Canada) and DNase (20 U/ml; Promega, Ottawa, Ontario, Canada). Tissue fragments were digested for three consecutive incubations of 50 min in a shaking water bath at 37 C. The tissue was dissociated after the first and third incubations by gentle aspiration in a 10-ml disposable sterile pipette and the tissues allowed to sediment to the bottom of the flask. At the end of the incubations, the cells were collected by centrifugation (34 x g) for 3 min, washed, and suspended in culture media [50 U/ml penicillin, 50 μg/ml streptomycin, 2 mM L-glutamine, 10 μg/ml insulin, 10 μg/ml transferrin, 80 ng/ml hydrocortisone, 1 μg/ml retinol, 10 ng/ml epidermal growth factor, 10 ng/ml cAMP, and 1% fetal bovine serum (FBS); Sigma-Aldrich]. The small aggregates of cells were then placed in 24-well plates and incubated in a humidified chamber at 32 C with 5% CO2. The culture media was changed every 24 h for 6 d.
Immortalization of epididymal cells
Epididymal cells (30–40% confluent) were transfected by calcium phosphate precipitation according to the method of Graham and Van der Eb (24). In a total aliquot of 2.5 μg DNA, 200 ng of a pBK-CMV plasmid containing the SV40 LTAg and neomycin resistance genes (25) were resuspended along with Sp64 plasmid (Promega Corp., Madison, WI) as carrier DNA in 25 μl nano-pure sterile water. The DNA solution was diluted with 25 μl sterile CaCl2 (4x; 2 mM Tris HCl, 0.2 mM EDTA, 500 mM CaCl2, pH 7.9). The mixture was then added drop by drop to 50 μl HBS (2x; 50 mM HEPES, 280 mM NaCl, 1.5 mM Na2HPO4-7H2O, pH 7.1), and a DNA-CaPO4 precipitate was formed by gently forming small air bubbles in the solution, followed by an incubation of 30 min at room temperature. The precipitate was then added dropwise directly onto the cultured cells (45 μl per well in a 24-well plate). The cells remained in contact with the precipitate for 24 h, the culture media was then changed, and the cells were cultured for another 24 h. Stable transfectants were selected by incubating the cells in media containing neomycin (200 μg/ml G418; GIBCO BRL, Burlington, Ontario, Canada) for 14 d. Resistant cells were then isolated by serial dilution and a stable cell line named RCE was generated. The initial culturing of the epididymal cells and the selection of stable clones were done in simple plastic culture wells; however, the cells divided very slowly under these conditions. To improve on the doubling time, the RCE cells were eventually replated in collagen-IV-coated culture dishes (BD Biosciences, Mississauga, Ontario, Canada). All subsequent experiments were then done on collagen-IV-coated plates with media containing 5% FBS and 5 nM testosterone. After 46 passages, the cells tested negative for mycoplasma contamination as determined by enzyme immunoassay (Roche Diagnostics GmbH, Mannheim, Germany)
Cell growth
RCE cells were plated at a density of 25,000 cells per well in 24-well culture plates coated with collagen IV. Cells were allowed to settle and adhere overnight. The next day, medium was changed, and this first time point was assigned as time zero. Medium was changed every 24 h before each time point (0, 24, 48, and 120 h), culture medium was removed from two of the wells and replaced with methylthiazolyldiphenyl-tetrazolium bromide (MTT) solution (Sigma-Aldrich; 0.5 mg/ml in culture medium) to measure cellular proliferation (26). Cells were then incubated for 2.5 h. The MTT solution was then removed, and the formazan crystals were solubilized with 1 ml dimethylsulfoxide per well. Three aliquots of 200 μl were placed in a 96-well plate and the absorbance at 570 was read using a microtiter plate reader (Power Wave X; Bio-Tek Instruments Inc., Winooski, VT)
Analyses of the cell cycle
RCE cells were trypsinized, recovered by centrifugation, and washed twice in PBS. Cells were fixed by resuspending the pellet in ice-cold ethanol and kept on ice for 30 min. The cells were then washed three times in PBS containing 2% FBS. The pellet was finally resuspended in a solution containing RNase A (100 μg/ml) and propidium iodide (50 μg/ml). DNA content of 1 x 106 cells per sample was analyzed using a FACScan apparatus (Becton Dickinson, Oakville, Ontario, Canada). Mouse spleen cells (a kind gift of Dr. J. Bernier, INRS) were used as control diploid cells.
Electron microscopy
RCE cells were incubated for 48 h and trypsinized (0.05% trypsin, 0.53 mM EDTA). The cells were then gently aspirated and transferred into a 15-ml tube. The cells were collected by centrifugation (300 x g) and resuspended in 2.5% glutaraldehyde in sodium cacodylate buffer for 24 h. The cells were then washed in PBS and postfixed in 1% ferrocyanide-reduced osmium tetroxide, dehydrated in ethanol and propylene oxide, and embedded in EPON. Thin sections (60 mm) were stained with uranyl acetate and lead citrate and examined for morphology with a Philips EM-400 electron microscope (Philips, Eindhoven, The Netherlands).
RT-PCR
Total cellular RNA was isolated using an Absolutely RNA RT-PCR miniprep kit (Stratagene, La Jolla, CA) according to the manufacturers’ instructions. Total RNA (500 ng) was reverse transcribed using an oligo (dT)16 primer. PCR was subsequently done to identify the presence of transcripts for SV40 LTAg, AR, ER, 4-ene-steroid-5-reductase (5R) types 1 and 2, rat epididymal-retinoic binding protein (rE-RABP), Crisp-1, occludin, claudin (Cldn)-1, -3, and -4, and connexin (Cx)-26, -32, -30.3, and -43. Primer sequences are listed in Table 1. PCR amplification was done using the following program: 94 C for 5 min, 30–40 cycles of 95 C for 30 sec; Tm for 30 sec, 94 C for 30 sec; 72 C for 5 min for the addition of poly-A overhangs and cooled to 4 C. For rE-RABP, Crisp-1, ER, and Cx32, a two-step PCR was performed using 5 min at 94 C, 30–40 cycles for 30 sec at 95 C, and melting temperature (Tm) for 1 min. PCR products were then separated on a 1.5% agarose gel and visualized with ethidium bromide using a Fluor-S Multi-Imager densitometer (Bio-Rad Laboratories, Mississauga, Ontario, Canada). Bands of the predicted size were excised, and the amplicons were extracted and cloned into plasmid. The modified plasmids were then amplified, purified, and sequenced. The identity of the amplified DNA was determined based on sequence homology with a BLAST search (GenBank). PCR were also done on RNA that was not reverse transcribed to assess whether or not the RNA contained any genomic DNA.
Androgen regulation of the gene
RCE cells (1.2 x 105) were seeded in six-well collagen-IV-coated plates. Cells were cultured in media containing 5 nM testosterone and 5% FBS. Cells were allowed to adhere to the culture dish overnight. The next day the cells were washed in PBS, and the culture medium was replaced with medium without testosterone containing 5% charcoal-stripped FBS. Cells were then treated with either ethanol (0.01%; control) or increasing doses of DHT (10 nM, 100 nM, and 1 μM in 0.01% final volume of ethanol) for 48 h. The medium was changed after 24 h. Data points for each experimental condition were done in triplicate, and the experiment was repeated once. At the end of the incubation period, cells were collected and total RNA was extracted using a commercial RNA extraction kit (Absolutely RNA RT-PCR miniprep kit; Stratagene). The RNA was reverse transcribed as described above and the androgen-responsive gene rE-RABP was amplified using 25 cycles with the rE-RABP primers and 30 cycles with the nested rE-RABP primers (Table 1). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. PCR products were separated by electrophoresis on an agarose gel and stained with ethidium bromide. The amplified DNA fragments on the gel were quantified by densitometry. The data were expressed relative to the average intensity of the control treatment group ± SEM (n = 6). To block androgen action, cells were treated with vehicle, flutamide (10 μM), DHT (1 μM), or DHT and flutamide (1 and 10 μM, respectively). Cells were exposed to medium containing 5% charcoal-stripped FBS and different doses of DHT for 48 h. At the end of the incubation, the cells were harvested and the RNA isolated and used for RT-PCR to amplify.
Overexpression of the rat AR and Western blot analysis
RCE cells were plated at a density of 250,000 cells per well in collagen-IV-coated six-well culture plates in complete medium. Cells were allowed to settle and adhere overnight. The next day, cells were washed with PBS and the medium was replaced by medium that did not contain antibiotics (1 ml per well). Cells were transfected using Lipofectamine 2000 reagent (Canadian Life Technologies) according to the manufacturer’s instructions, using a total of 4 μg DNA per well. Increasing amounts of pCMV-rAR expressing the rat AR (27) (kindly provided by Dr. F. French, University of North Carolina) was transfected into the cells, and pBlueScript was used both as a control and to normalize the DNA content of the transfection mixture. Twenty-four hours after transfection, the medium was removed and cells were washed in PBS and trypsinized. Pools of three wells were centrifuged and resuspended in 150 μl RIPA buffer (1x PBS, 1% IGEPAL, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with 10 μg/ml phenylmethylsulfonyl fluoride, 100 μM sodium orthovanadate, 1 nM DHT, and a protease inhibitor cocktail (Sigma). Cells were placed on ice for 20 min and then centrifuged at 10,000 x g for 10 min at 4 C to remove cellular debris. Protein concentrations in the cell extracts were determined using the Bradford method (Bradford reagent; Sigma). Total protein extracts of adult rat tissues were prepared in the same buffer and used as a control for AR detection.
Fifty micrograms of protein were separated by electrophoresis on a 7.5% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane (Bio-Rad Laboratories). The membrane was incubated overnight at 4 C in blocking buffer (5% dry milk and 0.1% Tween 20 in Tris-buffered saline) and subsequently for 2 h at room temperature in the same buffer containing 1 μg/ml of an anti-AR antibody (C-19; Santa Cruz Biotechnology, Santa Cruz, CA). Membranes were then probed with an alkaline phosphatase-conjugated goat antirabbit secondary antibody (0.4 μg/ml; Santa Cruz Biotechnology). The AR was revealed using a colorimetric kit (Bio-Rad Laboratories).
Cotransfections and luciferase activity measurement
To assess the functionality of the rat AR, transiently transfected into the RCE cells, cotransfections were done using the DNA vector expressing the rAR and a reporter plasmid containing the luciferase gene under the control of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) (28) (pMAMneo-LUC; kind gift from Dr. T. Schrader, Health Canada).
Cells were plated at a density of 50,000 cells per well in collagen-IV-coated 24-well culture plates in medium containing 5% charcoal-stripped FBS and without testosterone and hydrocortisone. Cells were allowed to settle and adhere overnight. The next day, cells were washed with PBS and the medium was replaced with the same medium but without antibiotics (500 μl/ml per well). Cells were cotransfected with an equal amount of the two plasmids (200 ng each per well) using Lipofectamine 2000. After 4 h, the medium was removed and cells were incubated with ethanol (0.01% in culture medium), testosterone (50 nM), or the synthetic androgens metribolone (50 nM; R1881) or mibolerone (50 nM). After a 24-h incubation, the medium was removed, the cells were washed with PBS, and total cellular proteins were extracted using the cell culture lysis buffer (Promega). Luciferase assays were performed using a luminometer (Dynex Technologies, Chantilly, VA) and the Promega luciferase assay reagents.
Immunofluorescent microscopy
To demonstrate that the RCE cell line was of epithelial origin, cells were immunostained with a polyclonal antikeratin antiserum raised against bovine epidermal keratin (85 μg/ml; Dako Corp., Carpinteria, CA). Immunofluorescent microscopy was also done to localize specific proteins normally present in epididymal junctional complexes. Experiments were done with commercially available antisera directed against occludin (Zymed Laboratories, South San Francisco, CA), Cx26 (Chemicon International, Temecula, CA), Cx32 (Chemicon), and Cx43 (Santa Cruz Biotechnology). The RCE cells were grown for 48 h on plastic chamber slides (Nalge Nunc International, Naperville, IL) coated with collagen IV (BD Biosciences). Cells were fixed in methanol at –20 C for 10 min and permeabilized in a solution of 0.3% Triton X-100 in PBS for 15 min at room temperature. Cells were then washed in 1x PBS and blocked in the same buffer containing 5% BSA and 0.01% NaN3 for 30 min at 37 C, followed by three 5-min washes in 1x PBS. Tissues were subsequently incubated with primary antibodies (occludin, 0.625 μg/ml; Cldn-1, 650 μg/ml; Cx26, 10 μg/ml; and Cx43, 4 μg/ml) for 90 min at room temperature, and washed three times for 5 min with 1x PBS. Tissues were then incubated with the appropriate secondary antibodies (FITC-conjugated antirabbit or antimouse, 7.5 μg/ml; Jackson ImmunoResearch Laboratories Ltd., West Grove, PA) for 45 min at 37 C. Cells were finally washed three times in PBS and mounted with Vectashield mounting medium containing propidium iodide (Vector Laboratories, Burlington, Ontario, Canada).
Results
Generation of a rat caput epididymal cell line (RCE)
Primary cultures of rat epididymal cells were transfected by calcium phosphate precipitation using 200 ng of expression plasmid coding for the SV40 LTAg and resistant to neomycin (G418) antibiotic. The efficiency of the transfections was assessed by transfecting cells with a plasmid expressing the luciferase gene as a reporter (data not shown). Transfected cells were selected by incubation with G418 in the culture media. By serial dilution, a single colony of resistant cells was obtained and further characterized. The immortalized epididymal cells were elongated in shape when nonconfluent and contained large nuclei. In contrast, confluent cells had a distinct ovoid shape (Fig. 1A). The cells were initially maintained on ordinary plastic; however, the cells showed sluggish growth on this support (doubling time > 4 d; data not shown). To improve on the cell growth characteristics of the epididymal cells, the cells were subsequently cultured on collagen-IV-coated plastic plates at 32 C. Under these conditions, the cells grew more rapidly with a doubling time of approximately 35 h (Fig. 1B). The cells were passaged twice weekly and have been continuously cultured for over 2 yr without incident. RT-PCR was used to confirm that the immortalized cells were positive for the SV40 LTAg. As expected, a major PCR product of 304 bp corresponding to the LTAg was amplified from these cells (data not shown). To assess the epithelial origin of the RCE cell line, a broad-spectrum antiserum was used to screen for cytokeratin (Fig. 1C). The prominent cytokeratin staining that we observed confirmed that our immortalized epididymal cells were indeed of epithelial origin (Fig. 1C, b). Cells incubated without primary antiserum were used as a negative control (Fig. 1C, a). Homogeneity of the cell population was confirmed by flow cytometry (Fig. 2). Mouse spleen cells were used as a control diploid cell population (Fig. 2A). RCE cells showed two peaks of fluorescence (Fig. 2B) corresponding to resting and dividing polyploidal cells. When the cells were combined, one could see that the fluorescence obtained from RCE cells did not overlap with the mouse spleen cells (diploid index = 1.800), indicating that the cells were tetraploid (Fig. 2C).
Ultrastructural analysis of RCE cells indicated that it is composed primarily of epithelial principal cells, although clear cells were also present. Of a total of 400 cells counted in various electron micrographs, approximately 1% of the total number of cells displayed characteristics associated with clear cells (five cells of a total of 415 cells). Narrow, apical, or basal cells were not present as were myoid cells or fibrocytes. RCE cells contained stereocilia on their cell surface, an irregular nucleus, and few endosomal elements but numerous mitochondria and free ribosomes (Fig. 3A). In addition to the Golgi apparatus, showing multiple stacks of saccules and associated coated and uncoated vesicles, numerous cisternae of rough and dilated endoplasmic reticulum were also observed. Junctional complexes were clearly apparent between adjacent cells (Fig. 3C). An occasional principal cell showed several large endosomes and lysosomes suggestive of principal cells of the intermediate zone of the caput epididymidis (Fig. 3D). Certain RCE cells contained few microvilli and an abundance of endosomal and lysosomal elements; however, these cells were scarce (Fig. 3B). Although isolated cells appeared polarized, with the nuclei localized at one pole of the cell and, at the opposing end, either the presence of microvilli in principal-like cells or an accumulation of endosomes and lysosomes in clear cells, electron microscopy analyses of cells grown on collagen-coated plates indicate that the cells were not completely polarized (Fig. 3E).
Expression of epididymal markers and androgen responsiveness
Because the androgen dependence of epididymal structure and function is well known, we next determined whether our RCE cell line still expressed the AR and could respond to androgen stimulation. First, RT-PCR was done with primers specific for the AR and other genes known to be expressed in the epididymis, including ER, 5R-1, 5R-2, and Crisp-1 (Figs. 4 and 5). A single PCR product of 524 bp was amplified with either RNA isolated from RCE cell lines or RNA from the intact caput region of the adult rat epididymis (Fig. 4). The identity of the PCR product as the AR was confirmed by sequencing. The levels of AR transcript in RCE cells was consistently low relative to levels in the epididymis. A similar approach was also used to detect the expression of ER (Fig. 4). Studies have shown that estradiol appears to be important in regulating the development of epithelial cells in the epididymis and in regulating ion exchange and fluid uptake from the lumen (29). Because the epididymis expresses a number of genes that are unique to this tissue, these genes then serve as useful markers for validating the epididymal origin of our cells. It is well characterized that testosterone is converted to DHT in the epididymis. In vivo studies have reported high concentrations of 5R-1 in the initial segment of the epididymis, and 5R-2 is present throughout the epididymis (4). In the present study, we observed that the RCE cell line expressed both 5R-1 and -2 (Fig. 4B). Two epididymal principal cell-specific genes were tested: Crisp-1 and rE-RABP (Fig. 5, A and B). Both genes were clearly expressed in RCE cells. Once again, RNA isolated from the intact caput region of the adult rat epididymis was used as positive control. All RNA samples were tested for genomic DNA contamination by PCR (data not shown).
To assess whether or not RCE were androgen responsive, we assessed the ability of DHT to stimulate rE-RABP, a well-known androgen-dependent epididymal gene (30). Cells cultured for 48 h in the presence of varying physiological doses of DHT showed an increase in mRNA levels (Fig. 6, top). An increase in rE-RABP expression occurred at 100 nM and 1 μM DHT. To assess whether or not we could block the DHT-stimulated increase in mRNA levels, cells were incubated with DHT (1 μM) and flutamide (10 μM). Both flutamide and DHT increased rE-RABP (Fig. 6, bottom). Simultaneous incubation of both DHT and flutamide did not result in any changes in the levels of rE-RABP. Therefore, although RCE exhibit many structural features of normal epididymal principal cells, they do not appear to be androgen responsive.
Because the RCE cell line displayed a limited response to androgen stimulation, we wanted to determine whether these cells could respond to low doses of androgens. Using an artificial cellular model in which cells were transfected with a vector expressing the rat AR, we were able to show that the AR could be overexpressed in RCE cells (Fig. 7). Furthermore, if the cells are cotransfected with an androgen-responsive vector containing androgen response elements linked to a luciferase gene, then it is possible to stimulate luciferase activity in a dose-response manner with low doses of testosterone ranging from 1–10 nM (Fig. 7B). At higher doses (10 nM to 1 μM), the response plateaued (data not shown). However, when cells were transfected with the MMTV-LTR vector with or without the AR and the cells challenged with either testosterone (50 nM) or equivalent concentrations of R1881 or mibolerone, only the cells transfected with the AR responded (Fig. 7C). This suggests that our cell line may not have sufficient levels of the AR or that there are other factors that we have yet to identify that are necessary to elicit a strong androgen response in the RCE cell line.
Expression of junctional proteins in RCE cells
Electron microscopy indicated that RCE cells formed contacts with one another resembling tight and gap junctions. To determine whether these cells expressed junctional proteins, we tested a variety of gap and tight junctional proteins that we have previously demonstrated to be present in the normal intact rat epididymis (6, 8, 11, 31). Using RT-PCR primers listed in Table 1, four different Cx were detected in RCE cells: Cx26, Cx30.3, Cx32, and Cx43 (Fig. 8I); the caput epididymidis from an intact adult rat was used as positive control. The sizes of the amplified products were identical to the predicted Cx sizes in all cases, and the identity of the PCR products was confirmed by sequencing and a BLAST homology search in GenBank. Transcripts for several tight junctional proteins (occludin, Cldn-1, Cldn-3, and Cldn-4) were also expressed in the RCE cell line. In all cases, the amplified PCR products were the same whether they were from RCE cells or the intact adult rat epididymis control (Fig. 8I).
To confirm that the transcripts for the different junctional proteins were functional and localized to the area of cell contacts, immunofluorescent microscopy was done for Cx26 and -43 as markers of gap junctions and for occludin and Cldn-1 as markers of tight junctions (Fig. 8II). As expected, Cx26 immunostaining was present between adjacent cells. The staining was punctate, indicating the presence of connexons, and these surrounded the entire periphery of the cells (Fig. 8II, A). Cx43 immunostaining was also present between adjacent cells and formed punctate structures around the entire cell (Fig. 8II, B). These results suggest that RCE cells, much like their in vivo epididymal counterparts, form functional gap junctions between each other. Using a similar approach, both occludin and Cldn-1 protein were clearly visible in RCE cell cultures (Fig. 8II, C). For both proteins, immunofluorescent staining was present along the plasma membrane where the cells make contact with each other (Fig. 8II, D). The immunolocalization pattern of these tight junctional proteins was similar to that reported for other cell types forming intercellular tight junctions (reviewed in Ref.31). Thus, in addition to gap junctions, the RCE cell line also appears to form intercellular tight junctions that are characteristic of in vivo epididymal epithelial principal cells.
Discussion
In the epididymis, complex cellular interactions between epithelial cells are essential for creating the specific microenvironment that is necessary for sperm to mature (acquire motility and the ability to fertilize eggs) as they traverse this duct (8). In addition, our previous work has established that intercellular junctions in the epididymis involve many proteins that are implicated either in gap junctional communication or the formation and maintenance of epididymal tight junctions that form the blood-epididymal barrier (8, 9, 32). To help define the roles of the different protein players involved in regulating epithelial cell-cell interactions and epithelial cell-sperm interactions in the epididymis, useful in vitro tools such as reliable cell lines in which epididymal gene expression can be modulated are needed. Unlike many other systems, however, these essential tools have not been readily available to epididymal physiologists. In the present study, we have generated the first stable epithelial cell line from the rat epididymis (RCE cell line) that retains many features of in vivo cells making it ideally suited for the study of epididymal functions.
RCE cells immortalized with the SV40 LTAg had an average doubling time of 35 h when maintained at 32 C, the normal physiological temperature of the rat epididymis. The growth characteristics of our cells are slower than those reported for canine epididymal cell lines that were also generated with the SV40 LTAg (20). The fact that our cells were maintained at a slightly lower temperature (32 C) may account for their longer doubling time. To date, these cells have been cultured for over 2 yr and their functions and composition have remained constant.
Electron microscopy revealed that our RCE cell line resembled epithelial principal cells, although some of the cells appeared to take on certain characteristic associated with clear cells (1% of total cells). Other cell types such as narrow, apical, basal, myoid, and fibrocytes were not evident. The presence of cells with similar characteristics as clear cells is interesting. The results of the flow cytometry data suggest that the cells in the culture are of a single type. For the different cell types to be present in the culture would have meant that there was incomplete separation of cells by serial dilution or immortalization of an undifferentiated epididymal cell that has differentiated into two distinct cell populations. The latter is unlikely, however, because few epithelial cells undergo active mitosis in the epithelium of the adult epididymis, and these cells are fully differentiated by postnatal d 39 (33). There is in fact very little information on the population of undifferentiated cells in the epididymis and whether environmental factors play a role in the differentiation of these cells into principal, clear, or other cell types (34, 35, 36). The fact that there remain cells that resemble clear cells even after many passages suggests that these cells may not be distinct cell types as one would have expected these to be eliminated with time. This is because if there had been two cell types at the time of separation, then one would expect that half the cells would be one type and the other half another type. The fact that these cells make up only 1% of the cells suggest that the cells divide at a different rate. If this were the case, it would mean that the slowly dividing cells would be eliminated over time, and this is not the case. It therefore appears likely that these cells develop a different phenotype in culture. More studies will be needed to determine the reason for these differences. Another interesting ultrastructural feature of RCE cells was the presence of large endosomes within certain cells (Fig. 3). In the intermediate zone of the epididymis, principal cells contain large endosomes (37). This suggests that some cells share features similar to principal cells of the intermediate zone, although such cells were less than 0.1% of total cells.
The morphology of single trypsinized cells was similar to those of polarized cells. Furthermore, these cells expressed both tight and adhering (data not shown) junctional proteins. Yet analysis of the cells on collagen-coated wells suggests that the cells are not completely polarized. Recently, Britan et al. (38) have reported that spontaneously immortalized mouse epididymal cells could be induced to polarize by modifying the culture condition and that epididymal genes would be expressed in the cells once they became polarized. This may not be the case for RCE cells, which appear to express many epididymal genes.
Transcripts for AR and ER were present in RCE cells, as were transcripts for both type 1 and type 2 5R, indicating that these cells have the capacity to respond to androgens and estrogens. However, the levels of AR were low relative to intact epididymis. Relative rE-RABP mRNA levels, a well-characterized androgen-dependent epididymal gene (30, 39), were higher in RCE cells treated with high epididymal concentrations of DHT (Fig. 6) (40) and only when using charcoal-stripped serum. However, the stimulation was not inhibited by flutamide treatment, suggesting that the stimulation is not the result of androgen action. However, using the MMTV-LTR vector, it is clear that the RCE cells do not respond to lower doses of testosterone unless the levels of the AR are increased. This suggests that the cells may not be androgen responsive, and this may be in part the result of low levels of the AR. It has been reported in other epididymal cell lines that the transformation of the cells reduces their responsiveness to testosterone (38, 41).
Direct intercellular communication is mediated by gap junctions, which form intercellular pores between adjacent cells. These pores are made up of radially aligned connexons from adjacent cells. These connexons are in turn made up of transmembrane proteins termed connexin (Cx) (42). We have previously shown that intercellular communication in the epididymis involves several Cx (Cx43, -26, -30.3, -31.1, and -32). The developmental patterns of Cx mRNA levels in the epididymis suggest that some Cx (e.g. Cx26 and Cx43) may play a role in the differentiation of the epididymal epithelium and that there is a switch from Cx26 to Cx31.1 during the development of the epididymis (43). Consistent with their in vivo principal cell counterparts, RCE cells strongly express many of the pertinent epididymal Cx, including Cx26, -30.3, -32, and -43 (Fig. 8).
One of the major functions of the epididymal epithelium is the formation of the blood-epididymal barrier. This barrier allows the composition of epididymal fluid to differ from the blood plasma by allowing receptor-mediated vectorial transport across the epithelium and preventing the loss of solutes and ions, thereby creating a specific microenvironment needed for sperm maturation (8, 9). The barrier also protects the maturing sperm from the immune system, because sperm are antigenic (44). Our studies have shown that epididymal tight junctions between principal cells are composed of occludin and at least seven different Cldn (Cldn-1, -3, -4, -6, -7, -8, and -16) (11, 45). As for the gap junctional proteins, many of these tight junctional proteins as well as tight junctions themselves were also expressed in RCE cells (Fig. 8), providing additional evidence that this cell line is highly representative of in vivo epididymal principal cells. This cell line model will, therefore, be particularly useful for studying many aspects of epididymal functions ranging from epididymal gene expression to epithelial cell-cell interactions and spermatozoa-epithelial cell interactions that are required for sperm to become mature.
Acknowledgments
We are grateful to Dr. D. W. Silversides (University of Montreal) for the gift of SV40 LTAg vector, Dr. F. French (University of North Carolina) for the rat androgen receptor expression vector, and T. Schrader (Health Canada) for the androgen-responsive MMTV-LTR vector. Dr. G. M. Cooke (Health Canada), J. Mui (McGill University), and M. Gregory (Institut National de la Recherche Scientifique) are thanked for their assistance and helpful suggestions. M. Pelletier and M. D’Elia (Institut National de Recherche et de Sécurité) are thanked for their assistance with the ploidy analyses.
Footnotes
This study was supported by the Armand-Frappier Foundation and the FQNRT (Quebec) in the form of studentships to N.S. and by a Discovery Grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada to D.G.C., an NSERC-Canadian Institutes for Health Research (CIHR) grant to L.H. and D.G.C., and a CIHR grant to R.V.
Abbreviations: AR, Androgen receptor; Cldn, claudin; Cx, connexin; DHT, dihydrotestosterone; ER, estrogen receptor-; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MMTV-LTR, mouse mammary tumor virus long terminal repeat; MTT, methylthiazolyldiphenyl-tetrazolium bromide; 5R, 4-ene-steroid-5-reductase; rE-RABP, epididymal retinoic acid binding protein; SV40 LTAg, simian virus 40 large T antigen; Tm, melting temperature.
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Ontogeny-Reproduction Research Unit (R.S.V.), Centre Hospitalier Universitaire Laval Research Centre and Centre de Recherche en Biologie de la Reproduction, Department of Obstetrics and Gynecology, Université Laval, Sainte-Foy, Québec, Canada G1K 7P4
Department of Anatomy and Cell Biology (L.H., D.G.C.), McGill University, Montreal, Québec, Canada H3A 2B2
Abstract
The epididymis is an androgen-dependent organ that allows spermatozoa to become fully functional as they pass through this tissue. The specialized functions of the epididymis are mediated by interactions between epididymal epithelial cells and between epididymal cells and spermatozoa. Although the critical role of the epididymis in sperm maturation is well established, the mechanisms regulating cell-cell interactions remain poorly understood because of the lack of appropriate cell line models. We now report the characterization of a novel rat caput epididymal cell line (RCE) that was immortalized by transfecting primary cultures of rat epididymal cells with the simian virus 40 large T antigen. At the electron microscope level, the cell line was composed of epithelial principal cells with characteristics of in vivo cells; principal cells had well-developed Golgi apparatus, abundant endoplasmic reticulum cisternae, and few endosomes. RCE cells expressed the mRNAs coding for the androgen receptor, estrogen receptor , and 4-ene-steroid-5--reductase types 1 and 2 as well as epididymal-specific markers Crisp-1 and epididymal retinoic acid binding protein. Epididymal retinoic acid binding protein expression was significantly induced with dihydrotestosterone, although this effect was not blocked by flutamide, suggesting that RCE cells are not androgen responsive. Neighboring cells formed tight and gap junctions characteristic of epididymal cells in vivo and expressed tight (occludin and claudin-1, -3, and -4) and gap junctional proteins (connexin-26, -30.3, -32, and -43). The RCE cell line displays many characteristics of epithelial principal cells, thus providing a model for studying epididymal cell functions.
Introduction
IN MAMMALS, TESTICULAR spermatozoa do not have the ability to fertilize or swim. The acquisition of these properties (sperm maturation) occurs as spermatozoa transit through the epididymis (1, 2, 3). The epididymis is an androgen-dependent organ whose structure and functions rely on hormones and other regulatory factors mainly secreted by the testes (4, 5). Morphologically, the epididymis can be subdivided into four distinct anatomical regions: the initial segment, the head (caput), the body (corpus), and the tail (cauda) (6). The complement of cell types that compose the epididymal epithelium varies according to the region being studied (1). The four major epididymal epithelial cell types have been referred to as principal, narrow, basal, and clear cells based on structural and functional parameters. Principal cells are by far the most abundant and are active in the transport and secretion of ions and small organic molecules, protein synthesis and secretion, and the absorption of fluid (1). The secretory products of these cells appear to be the major players in modifying spermatozoa and in the attainment of their maturational characteristics. The most active regulator of principal cell function is dihydrotestosterone (DHT), which is synthesized via the 5-reduction of testosterone occurring mainly in the initial segment (4, 5).
Cellular interactions in the epididymis are critical for the formation of a specialized blood-epididymal barrier between epithelial cells, and this involves mainly interactions between principal cells, the major epithelial cell type. This barrier creates a unique microenvironment in the epididymal lumen that is crucial for sperm maturation (7, 8, 9). In this way, ions, solutes, proteins, lipids, and androgen concentrations are markedly different from the blood plasma (8). Communication between epithelial cells is also believed to be important in coordinating epididymal function (7, 8, 9). In the epididymis, cellular interactions are under the influence of several endocrine and paracrine factors originating from the testis that ultimately regulate epididymal gene expression (10, 11). The complexity of these interactions is a strong indication that they are crucial for epididymal sperm maturation. Thus, demonstrating how direct cellular interactions in the epididymis are regulated and their significance to epididymal physiology remains a major but crucial challenge for our understanding of epididymal function and its role in sperm maturation.
Several in vivo and in vitro models have been employed to study epididymal functions. The majority of in vitro models have relied on the use of organ cultures, tubule cultures, and primary cell cultures (12, 13, 14, 15, 16, 17, 18). As a whole, these approaches have generated information primarily on the expression and regulation of epididymal secretory proteins. Because primary cultures of epididymal epithelial cells have a short half-life in vitro, and because these cells divide very slowly and can dedifferentiate, this approach has important limitations for the study of epididymal functions (19). Given the complexity of cell-cell interactions and varied epididymal functions within the epididymis, an immortalized epididymal cell line would therefore provide essential and invaluable tools for the study of these interactions and their contribution to epididymal functions.
A recent study by Telgmann et al. (20) reported the immortalization of canine epididymal cells by transfecting primary epididymal cultures with plasmids expressing either the simian virus 40 large T antigen (SV40 LTAg) or c-myc. The canine cell lines expressed the androgen receptor (AR), estrogen receptor- (ER), and the polyoma enhancer activator (PEA3) as well as the canine counterparts of the human HE-1, HE-4, and HE-5/CD52 genes (20). Cell lines have also recently been derived from the mouse epididymis. The first of these was generated from mice harboring a temperature-sensitive SV40 LTAg transgene (21). Much like the canine cells, these mouse epididymal cells also expressed the AR and some epididymal-specific proteins such as phosphatidylethanolamine-like binding protein, epididymal retinoic acid binding protein (E-RABP), and epididymal protein of 17 kDa (EP17) (21). Sipila et al. (22) also reported several mouse epididymal cell lines that were derived from transgenic animals expressing an SV40 LTAg transgene under the control of the glutathione peroxidase-5 promoter. Although many of these cells expressed AR, ER, and some epididymal proteins, few of the cell lines expressed more than two epididymal-specific proteins. The ultrastructure of these cells and how they compared with the normal mouse epididymis also remained unknown.
In the present study, we report the characterization of a novel rat caput epididymal cell line (named RCE) that was immortalized by transfecting primary cultures of rat epididymal cells with the SV40 LTAg. Importantly, the RCE cell line consists predominantly of principal cells that retain many characteristics, both structurally and functionally, of principal cells in vivo, making them ideally suited for studying epididymal cell function in relation to sperm maturation.
Materials and Methods
Primary epididymal cell cultures
Sprague-Dawley rats (40 d old; 151–175 g) were purchased from Charles River Canada Inc. (St. Constant, Québec, Canada). Rats were maintained under a constant photoperiod of 12 h light and 12 h dark and received food and water ad libitum. All animal protocols used in this study were approved by the University Animal Care Committee.
Rats were euthanized by CO2 asphyxiation, and epididymides were dissected from the animals under aseptic conditions and placed in DMEM/HAM’s F12 culture media containing penicillin (50 U/ml) and streptomycin (50 μg/ml) (Sigma-Aldrich, Mississauga, Ontario, Canada). The tissue was cleared of fatty materials, and the proximal region of the epididymis was retained (initial segment and caput epididymidis). Cells from these segments were isolated according to the methods of Kirchhoff et al. (23). Briefly, epididymides from four rats were finely cut with scissors. Tissue fragments (2–3 mm) were placed in media containing the antibiotics and collagenase (2 mg/ml; Life Technologies, Inc., Burlington, Ontario, Canada) and DNase (20 U/ml; Promega, Ottawa, Ontario, Canada). Tissue fragments were digested for three consecutive incubations of 50 min in a shaking water bath at 37 C. The tissue was dissociated after the first and third incubations by gentle aspiration in a 10-ml disposable sterile pipette and the tissues allowed to sediment to the bottom of the flask. At the end of the incubations, the cells were collected by centrifugation (34 x g) for 3 min, washed, and suspended in culture media [50 U/ml penicillin, 50 μg/ml streptomycin, 2 mM L-glutamine, 10 μg/ml insulin, 10 μg/ml transferrin, 80 ng/ml hydrocortisone, 1 μg/ml retinol, 10 ng/ml epidermal growth factor, 10 ng/ml cAMP, and 1% fetal bovine serum (FBS); Sigma-Aldrich]. The small aggregates of cells were then placed in 24-well plates and incubated in a humidified chamber at 32 C with 5% CO2. The culture media was changed every 24 h for 6 d.
Immortalization of epididymal cells
Epididymal cells (30–40% confluent) were transfected by calcium phosphate precipitation according to the method of Graham and Van der Eb (24). In a total aliquot of 2.5 μg DNA, 200 ng of a pBK-CMV plasmid containing the SV40 LTAg and neomycin resistance genes (25) were resuspended along with Sp64 plasmid (Promega Corp., Madison, WI) as carrier DNA in 25 μl nano-pure sterile water. The DNA solution was diluted with 25 μl sterile CaCl2 (4x; 2 mM Tris HCl, 0.2 mM EDTA, 500 mM CaCl2, pH 7.9). The mixture was then added drop by drop to 50 μl HBS (2x; 50 mM HEPES, 280 mM NaCl, 1.5 mM Na2HPO4-7H2O, pH 7.1), and a DNA-CaPO4 precipitate was formed by gently forming small air bubbles in the solution, followed by an incubation of 30 min at room temperature. The precipitate was then added dropwise directly onto the cultured cells (45 μl per well in a 24-well plate). The cells remained in contact with the precipitate for 24 h, the culture media was then changed, and the cells were cultured for another 24 h. Stable transfectants were selected by incubating the cells in media containing neomycin (200 μg/ml G418; GIBCO BRL, Burlington, Ontario, Canada) for 14 d. Resistant cells were then isolated by serial dilution and a stable cell line named RCE was generated. The initial culturing of the epididymal cells and the selection of stable clones were done in simple plastic culture wells; however, the cells divided very slowly under these conditions. To improve on the doubling time, the RCE cells were eventually replated in collagen-IV-coated culture dishes (BD Biosciences, Mississauga, Ontario, Canada). All subsequent experiments were then done on collagen-IV-coated plates with media containing 5% FBS and 5 nM testosterone. After 46 passages, the cells tested negative for mycoplasma contamination as determined by enzyme immunoassay (Roche Diagnostics GmbH, Mannheim, Germany)
Cell growth
RCE cells were plated at a density of 25,000 cells per well in 24-well culture plates coated with collagen IV. Cells were allowed to settle and adhere overnight. The next day, medium was changed, and this first time point was assigned as time zero. Medium was changed every 24 h before each time point (0, 24, 48, and 120 h), culture medium was removed from two of the wells and replaced with methylthiazolyldiphenyl-tetrazolium bromide (MTT) solution (Sigma-Aldrich; 0.5 mg/ml in culture medium) to measure cellular proliferation (26). Cells were then incubated for 2.5 h. The MTT solution was then removed, and the formazan crystals were solubilized with 1 ml dimethylsulfoxide per well. Three aliquots of 200 μl were placed in a 96-well plate and the absorbance at 570 was read using a microtiter plate reader (Power Wave X; Bio-Tek Instruments Inc., Winooski, VT)
Analyses of the cell cycle
RCE cells were trypsinized, recovered by centrifugation, and washed twice in PBS. Cells were fixed by resuspending the pellet in ice-cold ethanol and kept on ice for 30 min. The cells were then washed three times in PBS containing 2% FBS. The pellet was finally resuspended in a solution containing RNase A (100 μg/ml) and propidium iodide (50 μg/ml). DNA content of 1 x 106 cells per sample was analyzed using a FACScan apparatus (Becton Dickinson, Oakville, Ontario, Canada). Mouse spleen cells (a kind gift of Dr. J. Bernier, INRS) were used as control diploid cells.
Electron microscopy
RCE cells were incubated for 48 h and trypsinized (0.05% trypsin, 0.53 mM EDTA). The cells were then gently aspirated and transferred into a 15-ml tube. The cells were collected by centrifugation (300 x g) and resuspended in 2.5% glutaraldehyde in sodium cacodylate buffer for 24 h. The cells were then washed in PBS and postfixed in 1% ferrocyanide-reduced osmium tetroxide, dehydrated in ethanol and propylene oxide, and embedded in EPON. Thin sections (60 mm) were stained with uranyl acetate and lead citrate and examined for morphology with a Philips EM-400 electron microscope (Philips, Eindhoven, The Netherlands).
RT-PCR
Total cellular RNA was isolated using an Absolutely RNA RT-PCR miniprep kit (Stratagene, La Jolla, CA) according to the manufacturers’ instructions. Total RNA (500 ng) was reverse transcribed using an oligo (dT)16 primer. PCR was subsequently done to identify the presence of transcripts for SV40 LTAg, AR, ER, 4-ene-steroid-5-reductase (5R) types 1 and 2, rat epididymal-retinoic binding protein (rE-RABP), Crisp-1, occludin, claudin (Cldn)-1, -3, and -4, and connexin (Cx)-26, -32, -30.3, and -43. Primer sequences are listed in Table 1. PCR amplification was done using the following program: 94 C for 5 min, 30–40 cycles of 95 C for 30 sec; Tm for 30 sec, 94 C for 30 sec; 72 C for 5 min for the addition of poly-A overhangs and cooled to 4 C. For rE-RABP, Crisp-1, ER, and Cx32, a two-step PCR was performed using 5 min at 94 C, 30–40 cycles for 30 sec at 95 C, and melting temperature (Tm) for 1 min. PCR products were then separated on a 1.5% agarose gel and visualized with ethidium bromide using a Fluor-S Multi-Imager densitometer (Bio-Rad Laboratories, Mississauga, Ontario, Canada). Bands of the predicted size were excised, and the amplicons were extracted and cloned into plasmid. The modified plasmids were then amplified, purified, and sequenced. The identity of the amplified DNA was determined based on sequence homology with a BLAST search (GenBank). PCR were also done on RNA that was not reverse transcribed to assess whether or not the RNA contained any genomic DNA.
Androgen regulation of the gene
RCE cells (1.2 x 105) were seeded in six-well collagen-IV-coated plates. Cells were cultured in media containing 5 nM testosterone and 5% FBS. Cells were allowed to adhere to the culture dish overnight. The next day the cells were washed in PBS, and the culture medium was replaced with medium without testosterone containing 5% charcoal-stripped FBS. Cells were then treated with either ethanol (0.01%; control) or increasing doses of DHT (10 nM, 100 nM, and 1 μM in 0.01% final volume of ethanol) for 48 h. The medium was changed after 24 h. Data points for each experimental condition were done in triplicate, and the experiment was repeated once. At the end of the incubation period, cells were collected and total RNA was extracted using a commercial RNA extraction kit (Absolutely RNA RT-PCR miniprep kit; Stratagene). The RNA was reverse transcribed as described above and the androgen-responsive gene rE-RABP was amplified using 25 cycles with the rE-RABP primers and 30 cycles with the nested rE-RABP primers (Table 1). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. PCR products were separated by electrophoresis on an agarose gel and stained with ethidium bromide. The amplified DNA fragments on the gel were quantified by densitometry. The data were expressed relative to the average intensity of the control treatment group ± SEM (n = 6). To block androgen action, cells were treated with vehicle, flutamide (10 μM), DHT (1 μM), or DHT and flutamide (1 and 10 μM, respectively). Cells were exposed to medium containing 5% charcoal-stripped FBS and different doses of DHT for 48 h. At the end of the incubation, the cells were harvested and the RNA isolated and used for RT-PCR to amplify.
Overexpression of the rat AR and Western blot analysis
RCE cells were plated at a density of 250,000 cells per well in collagen-IV-coated six-well culture plates in complete medium. Cells were allowed to settle and adhere overnight. The next day, cells were washed with PBS and the medium was replaced by medium that did not contain antibiotics (1 ml per well). Cells were transfected using Lipofectamine 2000 reagent (Canadian Life Technologies) according to the manufacturer’s instructions, using a total of 4 μg DNA per well. Increasing amounts of pCMV-rAR expressing the rat AR (27) (kindly provided by Dr. F. French, University of North Carolina) was transfected into the cells, and pBlueScript was used both as a control and to normalize the DNA content of the transfection mixture. Twenty-four hours after transfection, the medium was removed and cells were washed in PBS and trypsinized. Pools of three wells were centrifuged and resuspended in 150 μl RIPA buffer (1x PBS, 1% IGEPAL, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with 10 μg/ml phenylmethylsulfonyl fluoride, 100 μM sodium orthovanadate, 1 nM DHT, and a protease inhibitor cocktail (Sigma). Cells were placed on ice for 20 min and then centrifuged at 10,000 x g for 10 min at 4 C to remove cellular debris. Protein concentrations in the cell extracts were determined using the Bradford method (Bradford reagent; Sigma). Total protein extracts of adult rat tissues were prepared in the same buffer and used as a control for AR detection.
Fifty micrograms of protein were separated by electrophoresis on a 7.5% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane (Bio-Rad Laboratories). The membrane was incubated overnight at 4 C in blocking buffer (5% dry milk and 0.1% Tween 20 in Tris-buffered saline) and subsequently for 2 h at room temperature in the same buffer containing 1 μg/ml of an anti-AR antibody (C-19; Santa Cruz Biotechnology, Santa Cruz, CA). Membranes were then probed with an alkaline phosphatase-conjugated goat antirabbit secondary antibody (0.4 μg/ml; Santa Cruz Biotechnology). The AR was revealed using a colorimetric kit (Bio-Rad Laboratories).
Cotransfections and luciferase activity measurement
To assess the functionality of the rat AR, transiently transfected into the RCE cells, cotransfections were done using the DNA vector expressing the rAR and a reporter plasmid containing the luciferase gene under the control of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) (28) (pMAMneo-LUC; kind gift from Dr. T. Schrader, Health Canada).
Cells were plated at a density of 50,000 cells per well in collagen-IV-coated 24-well culture plates in medium containing 5% charcoal-stripped FBS and without testosterone and hydrocortisone. Cells were allowed to settle and adhere overnight. The next day, cells were washed with PBS and the medium was replaced with the same medium but without antibiotics (500 μl/ml per well). Cells were cotransfected with an equal amount of the two plasmids (200 ng each per well) using Lipofectamine 2000. After 4 h, the medium was removed and cells were incubated with ethanol (0.01% in culture medium), testosterone (50 nM), or the synthetic androgens metribolone (50 nM; R1881) or mibolerone (50 nM). After a 24-h incubation, the medium was removed, the cells were washed with PBS, and total cellular proteins were extracted using the cell culture lysis buffer (Promega). Luciferase assays were performed using a luminometer (Dynex Technologies, Chantilly, VA) and the Promega luciferase assay reagents.
Immunofluorescent microscopy
To demonstrate that the RCE cell line was of epithelial origin, cells were immunostained with a polyclonal antikeratin antiserum raised against bovine epidermal keratin (85 μg/ml; Dako Corp., Carpinteria, CA). Immunofluorescent microscopy was also done to localize specific proteins normally present in epididymal junctional complexes. Experiments were done with commercially available antisera directed against occludin (Zymed Laboratories, South San Francisco, CA), Cx26 (Chemicon International, Temecula, CA), Cx32 (Chemicon), and Cx43 (Santa Cruz Biotechnology). The RCE cells were grown for 48 h on plastic chamber slides (Nalge Nunc International, Naperville, IL) coated with collagen IV (BD Biosciences). Cells were fixed in methanol at –20 C for 10 min and permeabilized in a solution of 0.3% Triton X-100 in PBS for 15 min at room temperature. Cells were then washed in 1x PBS and blocked in the same buffer containing 5% BSA and 0.01% NaN3 for 30 min at 37 C, followed by three 5-min washes in 1x PBS. Tissues were subsequently incubated with primary antibodies (occludin, 0.625 μg/ml; Cldn-1, 650 μg/ml; Cx26, 10 μg/ml; and Cx43, 4 μg/ml) for 90 min at room temperature, and washed three times for 5 min with 1x PBS. Tissues were then incubated with the appropriate secondary antibodies (FITC-conjugated antirabbit or antimouse, 7.5 μg/ml; Jackson ImmunoResearch Laboratories Ltd., West Grove, PA) for 45 min at 37 C. Cells were finally washed three times in PBS and mounted with Vectashield mounting medium containing propidium iodide (Vector Laboratories, Burlington, Ontario, Canada).
Results
Generation of a rat caput epididymal cell line (RCE)
Primary cultures of rat epididymal cells were transfected by calcium phosphate precipitation using 200 ng of expression plasmid coding for the SV40 LTAg and resistant to neomycin (G418) antibiotic. The efficiency of the transfections was assessed by transfecting cells with a plasmid expressing the luciferase gene as a reporter (data not shown). Transfected cells were selected by incubation with G418 in the culture media. By serial dilution, a single colony of resistant cells was obtained and further characterized. The immortalized epididymal cells were elongated in shape when nonconfluent and contained large nuclei. In contrast, confluent cells had a distinct ovoid shape (Fig. 1A). The cells were initially maintained on ordinary plastic; however, the cells showed sluggish growth on this support (doubling time > 4 d; data not shown). To improve on the cell growth characteristics of the epididymal cells, the cells were subsequently cultured on collagen-IV-coated plastic plates at 32 C. Under these conditions, the cells grew more rapidly with a doubling time of approximately 35 h (Fig. 1B). The cells were passaged twice weekly and have been continuously cultured for over 2 yr without incident. RT-PCR was used to confirm that the immortalized cells were positive for the SV40 LTAg. As expected, a major PCR product of 304 bp corresponding to the LTAg was amplified from these cells (data not shown). To assess the epithelial origin of the RCE cell line, a broad-spectrum antiserum was used to screen for cytokeratin (Fig. 1C). The prominent cytokeratin staining that we observed confirmed that our immortalized epididymal cells were indeed of epithelial origin (Fig. 1C, b). Cells incubated without primary antiserum were used as a negative control (Fig. 1C, a). Homogeneity of the cell population was confirmed by flow cytometry (Fig. 2). Mouse spleen cells were used as a control diploid cell population (Fig. 2A). RCE cells showed two peaks of fluorescence (Fig. 2B) corresponding to resting and dividing polyploidal cells. When the cells were combined, one could see that the fluorescence obtained from RCE cells did not overlap with the mouse spleen cells (diploid index = 1.800), indicating that the cells were tetraploid (Fig. 2C).
Ultrastructural analysis of RCE cells indicated that it is composed primarily of epithelial principal cells, although clear cells were also present. Of a total of 400 cells counted in various electron micrographs, approximately 1% of the total number of cells displayed characteristics associated with clear cells (five cells of a total of 415 cells). Narrow, apical, or basal cells were not present as were myoid cells or fibrocytes. RCE cells contained stereocilia on their cell surface, an irregular nucleus, and few endosomal elements but numerous mitochondria and free ribosomes (Fig. 3A). In addition to the Golgi apparatus, showing multiple stacks of saccules and associated coated and uncoated vesicles, numerous cisternae of rough and dilated endoplasmic reticulum were also observed. Junctional complexes were clearly apparent between adjacent cells (Fig. 3C). An occasional principal cell showed several large endosomes and lysosomes suggestive of principal cells of the intermediate zone of the caput epididymidis (Fig. 3D). Certain RCE cells contained few microvilli and an abundance of endosomal and lysosomal elements; however, these cells were scarce (Fig. 3B). Although isolated cells appeared polarized, with the nuclei localized at one pole of the cell and, at the opposing end, either the presence of microvilli in principal-like cells or an accumulation of endosomes and lysosomes in clear cells, electron microscopy analyses of cells grown on collagen-coated plates indicate that the cells were not completely polarized (Fig. 3E).
Expression of epididymal markers and androgen responsiveness
Because the androgen dependence of epididymal structure and function is well known, we next determined whether our RCE cell line still expressed the AR and could respond to androgen stimulation. First, RT-PCR was done with primers specific for the AR and other genes known to be expressed in the epididymis, including ER, 5R-1, 5R-2, and Crisp-1 (Figs. 4 and 5). A single PCR product of 524 bp was amplified with either RNA isolated from RCE cell lines or RNA from the intact caput region of the adult rat epididymis (Fig. 4). The identity of the PCR product as the AR was confirmed by sequencing. The levels of AR transcript in RCE cells was consistently low relative to levels in the epididymis. A similar approach was also used to detect the expression of ER (Fig. 4). Studies have shown that estradiol appears to be important in regulating the development of epithelial cells in the epididymis and in regulating ion exchange and fluid uptake from the lumen (29). Because the epididymis expresses a number of genes that are unique to this tissue, these genes then serve as useful markers for validating the epididymal origin of our cells. It is well characterized that testosterone is converted to DHT in the epididymis. In vivo studies have reported high concentrations of 5R-1 in the initial segment of the epididymis, and 5R-2 is present throughout the epididymis (4). In the present study, we observed that the RCE cell line expressed both 5R-1 and -2 (Fig. 4B). Two epididymal principal cell-specific genes were tested: Crisp-1 and rE-RABP (Fig. 5, A and B). Both genes were clearly expressed in RCE cells. Once again, RNA isolated from the intact caput region of the adult rat epididymis was used as positive control. All RNA samples were tested for genomic DNA contamination by PCR (data not shown).
To assess whether or not RCE were androgen responsive, we assessed the ability of DHT to stimulate rE-RABP, a well-known androgen-dependent epididymal gene (30). Cells cultured for 48 h in the presence of varying physiological doses of DHT showed an increase in mRNA levels (Fig. 6, top). An increase in rE-RABP expression occurred at 100 nM and 1 μM DHT. To assess whether or not we could block the DHT-stimulated increase in mRNA levels, cells were incubated with DHT (1 μM) and flutamide (10 μM). Both flutamide and DHT increased rE-RABP (Fig. 6, bottom). Simultaneous incubation of both DHT and flutamide did not result in any changes in the levels of rE-RABP. Therefore, although RCE exhibit many structural features of normal epididymal principal cells, they do not appear to be androgen responsive.
Because the RCE cell line displayed a limited response to androgen stimulation, we wanted to determine whether these cells could respond to low doses of androgens. Using an artificial cellular model in which cells were transfected with a vector expressing the rat AR, we were able to show that the AR could be overexpressed in RCE cells (Fig. 7). Furthermore, if the cells are cotransfected with an androgen-responsive vector containing androgen response elements linked to a luciferase gene, then it is possible to stimulate luciferase activity in a dose-response manner with low doses of testosterone ranging from 1–10 nM (Fig. 7B). At higher doses (10 nM to 1 μM), the response plateaued (data not shown). However, when cells were transfected with the MMTV-LTR vector with or without the AR and the cells challenged with either testosterone (50 nM) or equivalent concentrations of R1881 or mibolerone, only the cells transfected with the AR responded (Fig. 7C). This suggests that our cell line may not have sufficient levels of the AR or that there are other factors that we have yet to identify that are necessary to elicit a strong androgen response in the RCE cell line.
Expression of junctional proteins in RCE cells
Electron microscopy indicated that RCE cells formed contacts with one another resembling tight and gap junctions. To determine whether these cells expressed junctional proteins, we tested a variety of gap and tight junctional proteins that we have previously demonstrated to be present in the normal intact rat epididymis (6, 8, 11, 31). Using RT-PCR primers listed in Table 1, four different Cx were detected in RCE cells: Cx26, Cx30.3, Cx32, and Cx43 (Fig. 8I); the caput epididymidis from an intact adult rat was used as positive control. The sizes of the amplified products were identical to the predicted Cx sizes in all cases, and the identity of the PCR products was confirmed by sequencing and a BLAST homology search in GenBank. Transcripts for several tight junctional proteins (occludin, Cldn-1, Cldn-3, and Cldn-4) were also expressed in the RCE cell line. In all cases, the amplified PCR products were the same whether they were from RCE cells or the intact adult rat epididymis control (Fig. 8I).
To confirm that the transcripts for the different junctional proteins were functional and localized to the area of cell contacts, immunofluorescent microscopy was done for Cx26 and -43 as markers of gap junctions and for occludin and Cldn-1 as markers of tight junctions (Fig. 8II). As expected, Cx26 immunostaining was present between adjacent cells. The staining was punctate, indicating the presence of connexons, and these surrounded the entire periphery of the cells (Fig. 8II, A). Cx43 immunostaining was also present between adjacent cells and formed punctate structures around the entire cell (Fig. 8II, B). These results suggest that RCE cells, much like their in vivo epididymal counterparts, form functional gap junctions between each other. Using a similar approach, both occludin and Cldn-1 protein were clearly visible in RCE cell cultures (Fig. 8II, C). For both proteins, immunofluorescent staining was present along the plasma membrane where the cells make contact with each other (Fig. 8II, D). The immunolocalization pattern of these tight junctional proteins was similar to that reported for other cell types forming intercellular tight junctions (reviewed in Ref.31). Thus, in addition to gap junctions, the RCE cell line also appears to form intercellular tight junctions that are characteristic of in vivo epididymal epithelial principal cells.
Discussion
In the epididymis, complex cellular interactions between epithelial cells are essential for creating the specific microenvironment that is necessary for sperm to mature (acquire motility and the ability to fertilize eggs) as they traverse this duct (8). In addition, our previous work has established that intercellular junctions in the epididymis involve many proteins that are implicated either in gap junctional communication or the formation and maintenance of epididymal tight junctions that form the blood-epididymal barrier (8, 9, 32). To help define the roles of the different protein players involved in regulating epithelial cell-cell interactions and epithelial cell-sperm interactions in the epididymis, useful in vitro tools such as reliable cell lines in which epididymal gene expression can be modulated are needed. Unlike many other systems, however, these essential tools have not been readily available to epididymal physiologists. In the present study, we have generated the first stable epithelial cell line from the rat epididymis (RCE cell line) that retains many features of in vivo cells making it ideally suited for the study of epididymal functions.
RCE cells immortalized with the SV40 LTAg had an average doubling time of 35 h when maintained at 32 C, the normal physiological temperature of the rat epididymis. The growth characteristics of our cells are slower than those reported for canine epididymal cell lines that were also generated with the SV40 LTAg (20). The fact that our cells were maintained at a slightly lower temperature (32 C) may account for their longer doubling time. To date, these cells have been cultured for over 2 yr and their functions and composition have remained constant.
Electron microscopy revealed that our RCE cell line resembled epithelial principal cells, although some of the cells appeared to take on certain characteristic associated with clear cells (1% of total cells). Other cell types such as narrow, apical, basal, myoid, and fibrocytes were not evident. The presence of cells with similar characteristics as clear cells is interesting. The results of the flow cytometry data suggest that the cells in the culture are of a single type. For the different cell types to be present in the culture would have meant that there was incomplete separation of cells by serial dilution or immortalization of an undifferentiated epididymal cell that has differentiated into two distinct cell populations. The latter is unlikely, however, because few epithelial cells undergo active mitosis in the epithelium of the adult epididymis, and these cells are fully differentiated by postnatal d 39 (33). There is in fact very little information on the population of undifferentiated cells in the epididymis and whether environmental factors play a role in the differentiation of these cells into principal, clear, or other cell types (34, 35, 36). The fact that there remain cells that resemble clear cells even after many passages suggests that these cells may not be distinct cell types as one would have expected these to be eliminated with time. This is because if there had been two cell types at the time of separation, then one would expect that half the cells would be one type and the other half another type. The fact that these cells make up only 1% of the cells suggest that the cells divide at a different rate. If this were the case, it would mean that the slowly dividing cells would be eliminated over time, and this is not the case. It therefore appears likely that these cells develop a different phenotype in culture. More studies will be needed to determine the reason for these differences. Another interesting ultrastructural feature of RCE cells was the presence of large endosomes within certain cells (Fig. 3). In the intermediate zone of the epididymis, principal cells contain large endosomes (37). This suggests that some cells share features similar to principal cells of the intermediate zone, although such cells were less than 0.1% of total cells.
The morphology of single trypsinized cells was similar to those of polarized cells. Furthermore, these cells expressed both tight and adhering (data not shown) junctional proteins. Yet analysis of the cells on collagen-coated wells suggests that the cells are not completely polarized. Recently, Britan et al. (38) have reported that spontaneously immortalized mouse epididymal cells could be induced to polarize by modifying the culture condition and that epididymal genes would be expressed in the cells once they became polarized. This may not be the case for RCE cells, which appear to express many epididymal genes.
Transcripts for AR and ER were present in RCE cells, as were transcripts for both type 1 and type 2 5R, indicating that these cells have the capacity to respond to androgens and estrogens. However, the levels of AR were low relative to intact epididymis. Relative rE-RABP mRNA levels, a well-characterized androgen-dependent epididymal gene (30, 39), were higher in RCE cells treated with high epididymal concentrations of DHT (Fig. 6) (40) and only when using charcoal-stripped serum. However, the stimulation was not inhibited by flutamide treatment, suggesting that the stimulation is not the result of androgen action. However, using the MMTV-LTR vector, it is clear that the RCE cells do not respond to lower doses of testosterone unless the levels of the AR are increased. This suggests that the cells may not be androgen responsive, and this may be in part the result of low levels of the AR. It has been reported in other epididymal cell lines that the transformation of the cells reduces their responsiveness to testosterone (38, 41).
Direct intercellular communication is mediated by gap junctions, which form intercellular pores between adjacent cells. These pores are made up of radially aligned connexons from adjacent cells. These connexons are in turn made up of transmembrane proteins termed connexin (Cx) (42). We have previously shown that intercellular communication in the epididymis involves several Cx (Cx43, -26, -30.3, -31.1, and -32). The developmental patterns of Cx mRNA levels in the epididymis suggest that some Cx (e.g. Cx26 and Cx43) may play a role in the differentiation of the epididymal epithelium and that there is a switch from Cx26 to Cx31.1 during the development of the epididymis (43). Consistent with their in vivo principal cell counterparts, RCE cells strongly express many of the pertinent epididymal Cx, including Cx26, -30.3, -32, and -43 (Fig. 8).
One of the major functions of the epididymal epithelium is the formation of the blood-epididymal barrier. This barrier allows the composition of epididymal fluid to differ from the blood plasma by allowing receptor-mediated vectorial transport across the epithelium and preventing the loss of solutes and ions, thereby creating a specific microenvironment needed for sperm maturation (8, 9). The barrier also protects the maturing sperm from the immune system, because sperm are antigenic (44). Our studies have shown that epididymal tight junctions between principal cells are composed of occludin and at least seven different Cldn (Cldn-1, -3, -4, -6, -7, -8, and -16) (11, 45). As for the gap junctional proteins, many of these tight junctional proteins as well as tight junctions themselves were also expressed in RCE cells (Fig. 8), providing additional evidence that this cell line is highly representative of in vivo epididymal principal cells. This cell line model will, therefore, be particularly useful for studying many aspects of epididymal functions ranging from epididymal gene expression to epithelial cell-cell interactions and spermatozoa-epithelial cell interactions that are required for sperm to become mature.
Acknowledgments
We are grateful to Dr. D. W. Silversides (University of Montreal) for the gift of SV40 LTAg vector, Dr. F. French (University of North Carolina) for the rat androgen receptor expression vector, and T. Schrader (Health Canada) for the androgen-responsive MMTV-LTR vector. Dr. G. M. Cooke (Health Canada), J. Mui (McGill University), and M. Gregory (Institut National de la Recherche Scientifique) are thanked for their assistance and helpful suggestions. M. Pelletier and M. D’Elia (Institut National de Recherche et de Sécurité) are thanked for their assistance with the ploidy analyses.
Footnotes
This study was supported by the Armand-Frappier Foundation and the FQNRT (Quebec) in the form of studentships to N.S. and by a Discovery Grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada to D.G.C., an NSERC-Canadian Institutes for Health Research (CIHR) grant to L.H. and D.G.C., and a CIHR grant to R.V.
Abbreviations: AR, Androgen receptor; Cldn, claudin; Cx, connexin; DHT, dihydrotestosterone; ER, estrogen receptor-; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MMTV-LTR, mouse mammary tumor virus long terminal repeat; MTT, methylthiazolyldiphenyl-tetrazolium bromide; 5R, 4-ene-steroid-5-reductase; rE-RABP, epididymal retinoic acid binding protein; SV40 LTAg, simian virus 40 large T antigen; Tm, melting temperature.
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