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In vitro culture of epithelial cells from the caput, corpus, and cauda epididymis of Sus domesticus
Theriogenology 62 (2004) 929–942
In vitro culture of epithelial cells from the caput, corpus, and cauda epididymis of Sus domesticus Judit Bassols*, Elisabeth Ka´da´r, M Dolors Briz, Elisabeth Pinart, Sı´lvia Sancho, Nu´ria Garcia-Gil, Elena Badia, Anna Pruneda, Eva Bussalleu, Marc Yeste, Sergi Bonet Biotechnology of Porcine Reproduction, Department of Biology, Faculty of Sciences, University of Girona, Campus Montilivi sn, 17071 Girona, Spain Received 6 August 2003; received in revised form 10 December 2003; accepted 14 December 2003
Abstract This work describes a protocol to culture epididymal epithelial cells from the caput, corpus, and cauda regions of Sus domesticus. Epididymal epithelial fragments were obtained by dissection and enzymatic digestion with collagenase. About 30 epididymal fragments from each epididymal region were cultured in 24-well culture plates with supplemented RPMI-1640 medium at 37 8C, 5% CO2 in air, and 100% humidity. A confluent monolayer of polygonal and tightly packed epithelioid cells from the three epididymal regions was obtained after 12–16 days in culture and maintained in vitro for more than 60 days. The proportion of epididymal epithelial cells in these cultures was assessed by immunofluorescent staining for cytokeratins. Throughout the 2 months of culture, about 80% of the cells were cytokeratin-positive. Electron microscopy observations indicated that cultured cells from caput, corpus, and cauda epididymal regions were tightly adhered to each other by junctional complexes and that stereocilia were present in their apical membranes. Moreover, the presence of an extensive rough endoplasmic reticulum, Golgi apparatus and numerous vesicles in the cytoplasm suggested that cultured cells maintained secretory and absorptive activities. These results show that the epididymal epithelial cells in culture from S. domesticus retain some fundamental features that characterize the epididymal epithelium in the intact organ. This system might be a valuable tool for studying the mechanism of sperm maturation in vitro, including epididymal cell secretions and the analysis of regional differences. # 2004 Elsevier Inc. All rights reserved. Keywords: Boar; Sus domesticus; Epididymis; Epithelium; Epithelial cells; In vitro culture; Ultrastructure
* Corresponding author. Tel.: þ34-972418366; fax: þ34-972418150. E-mail address: [email protected] (J. Bassols).
0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2003.12.015
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1. Introduction It has been demonstrated that, in mammals, the epididymis plays an essential role in sperm maturation and storage [1]. Sperm maturation, which takes place during the transit of spermatozoa through the different epididymal regions [2], is a critical process in the acquisition of sperm motility and fertilizing ability. Functional changes during this process are the result of interaction between spermatozoa and epididymal epithelial cells, which need to be mediated by sequential changes in the protein composition of the epididymal fluid [3]. Different methods has been used to purify and identify many epididymal secreted proteins, but their role and importance in sperm physiology still needs to be established more fully [4–6]. Moreover, most of these methods make it difficult to study the activities of the epididymal epithelium without interference of testicular and non-epithelial tissue factors. To overcome this limitation, primary cultures of epididymal epithelial cells have been devised in different animal models. These in vitro cultures provide an attractive and simplified model system for the analysis of epithelial cell metabolic activities and their regulation because the synthesis and secretion of specific components can be monitored under defined conditions. In addition, data related to sperm maturation are provided by coculture experiments where intimate interactions between spermatozoa and epididymal epithelial cells can be examined in detail [16,23,24]. Some of the protocols employed to culture epididymal epithelial cells involve the isolation of a purified suspension of single epithelial cells [7–12]. However, the disruption of the epithelial architecture and the lack of basal lamina and other peritubular elements compromise the cells, which rapidly de-differentiate and lose function in culture. Currently, most methods use fragments or plaques of epithelium prepared after enzyme digestion [13–16]. Although differences in the enzymatic digestion procedure and the culture medium used have been observed, these protocols can generate cell monolayers in which epididymal epithelial cells retain their morphological characteristics, remain polarized and maintain absorptive and secretory activities, mimicking in vivo conditions [17,18]. Different studies have demonstrated that cultured epididymal epithelial cells can metabolize testosterone to the active metabolite, dihydrotestosterone [15] and synthesize and secrete alkaline phosphatase, acid phosphatase and N-acetylglucosaminidase [17,20]. Other secretory proteins, such as glycosidases [21] and glutathione-related enzymes [22] have also been described in epididymal cell cultures. In addition, an increase in progressive sperm motility and enhanced sperm survival has been shown in coculture with epididymal epithelial cells [13,14]. Moreover, it has been observed that several maturation-related compounds expressed by epididymal epithelium bind to spermatozoa in co-culture [23,24]. The aim of this study was to develop a cell culture system capable of supporting epithelial cells from caput, corpus, and cauda epididymis of Sus domesticus. This system will be used to identify and characterize boar epithelial cell secretions in specific regions of the epididymis. It will also be used for coculture with immature spermatozoa in order to study the sperm maturation process and the specific interactions between spermatozoa and epididymal epithelial cells in boars.
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2. Materials and methods 2.1. Animals Epididymal tissue was obtained from Pietrain and Iberian boars of 8–12 months of age. Once the animals were slaughtered, the testis and epididymis were located, transferred to sterile PBS (Gibco, Invitrogen S.A., Barcelona, Spain), supplemented with 50 U ml1 penicillin G and 50 mg ml1 streptomycin (Gibco, Invitrogen S.A.) and brought to the laboratory within 45–60 min. The boars were slaughtered by a veterinarian following approved guidelines for the ethical treatment of animals. 2.2. Tissue isolation and cell culture The epididymidis were removed from the testis and separated into three different segments: caput, corpus and cauda. Each epididymal segment was placed in a 150 mm culture dish (Nunc, LabClinics, Barcelona, Spain), covered with supplemented PBS and minced into fragments (2–5 mm in length). After three washes with supplemented PBS, tubule fragments were transferred into supplemented PBS containing 300 U ml1 collagenase (type VII, Sigma, Madrid, Spain) and incubated at 37 8C in a water bath for 2 h (in the case of the caput and corpus fragments) or 3 h (in the case of the cauda fragments). After incubation, tubule fragments (settled by gravity) were washed in three changes of the culture medium (see below). The supernatant was discarded and the pellet was resuspended with culture medium containing 150 U ml1 collagenase. Tubule fragments of caput, corpus, and cauda regions were incubated at 37 8C in a water bath, for 1, 2, and 3 h, respectively. After washing in culture medium, three times for 5 min at 200 g, usually about 30 tubule fragments were transferred to each well of a 24-well culture plate (Nunc, LabClinics) in 1 ml fresh culture medium and incubated at 37 8C in 5% CO2 in air and 100% humidity. After the first 24 h and then every 48 h, about half of the medium was changed during the first six days of culture. From then on, 90% of the medium was replaced every 48 h. Culture samples prepared for electron microscopy were treated as described above, except that 15 tubule fragments were plated into culture plate polycarbonate inserts (12 mm in diameter, 0.4 mm pore size; Millipore Ibe´ rica S.A., Madrid, Spain) in 500 ml culture medium. The inserts were then placed in a 24-well culture plate in 1 ml fresh culture medium. For immunofluorescence, 30 tubule fragments were plated into 0.1% gelatin (Merck, Madrid, Spain) pre-coated cover slips into a 24-well culture plate in 1 ml fresh culture medium. 2.3. Culture medium preparation The medium used for boar epididymal cell cultures was RPMI medium (Gibco, Invitrogen S.A.) supplemented with 10% of fetal calf serum (FCS) (PAA Laboratories, LabClinics), 1 mM D-sodium pyruvate (Gibco, Invitrogen S.A.), 100 nM insulin (Sigma), 200 nM hydrocortisone (Sigma), 200 nM testosterone (Fluka, Madrid, Spain), 1 mM dihydrotestosterone (Fluka), 5 mg ml1 transferrin (Sigma), 1 mg ml1 retinol acetate
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(Sigma), 25 mM Hepes (Gibco, Invitrogen S.A.), 50 U ml1 of penicillin G and 50 mg ml1 streptomycin (Gibco, Invitrogen S.A.). The culture medium was filtered with 0.2 mm filters (Pall-Gelman Laboratory, An Arbor, USA) and stored at 4 8C. 2.4. Immunofluorescence The percentages of epididymal epithelial cells in culture were assessed by immunofluorescent localization using a cytokeratin monoclonal antibody. At 14, 21, 28, 35, 42, and 49 days in culture, cells growing on 0.1% gelatin pre-coated cover slips were fixed with 3% p-formaldehyde (Merck) and 60 mM saccharose (Merck) in 0.1 M PBS (Merck) for 30 min. After three 10-min washes with 10 mM PBS, the cells were permeabilized for 10 min in a solution of 10 mM PBS containing 0.1% Triton X-100 (Sigma). Then cells were rinsed in 10 mM PBS for 5 min and incubated for 10 min in a blocking solution consisting of 10 mM PBS with 20 mM glycine (Serva, Innogenetics, Gent, Belgium) and 1% BSA (Sigma). The cells were stained with a mouse anti-pan cytokeratin monoclonal antibody (Chemicon, Pacisa-Giralt, Barcelona, Spain) (1:20) for 45 min at 37 8C, rinsed twice in PBS for 10 min and incubated in a 1:50 dilution of the rabbit anti-mouse immunoglobulin antibody conjugate FITC (Dako Diagnosticos S.A., Barcelona, Spain) for 30 min at 37 8C. After two 10-min washes in PBS, the cells were stained with 5 mM bisbenzimide (Sigma) for 7 min to localize the nucleus. Finally, the cells on the cover slips were covered with mounting medium (Sigma) and examined under a Leica DMR-XA fluorescence microscope. The percentages of cytokeratin-positive cells were determined by flow cytometry (FACSCalibur, Becton Dickinson). In order to provide control samples for background fluorescence, cells were incubated with a primary irrelevant monoclonal antibody (Chemicon, Pacisa-Giralt). 2.5. Light microscopy Cells in the culture plates were examined every other day using an inverted phasecontrast microscope (Zeiss, Telaval 31) and photographs were taken through a Ricoh, XRX 3000 camera. The extent of the surface covered by cells (percentage of confluence) and morphological characteristics of cultured cells were performed on different days of culture. Cell concentration and viability was also determined using trypan blue exclusion test. 2.6. Electron microscopy Cells grown on polycarbonate cell culture inserts were recovered at 7, 14, 21, 28, and 35 days in culture and fixed with 2.5% glutaraldehyde (Electron Microscopy Sciences, Aname, Barcelona, Spain) in 0.1 M cacodylate buffer (Agar Scientific LTD, Leica, Barcelona, Spain) for 1 h at 4 8C. The cells were rinsed in 0.1 M cacodylate buffer and post-fixed in 0.1 M cacodylate buffer containing 0.1% OsO4 (Electron Microscopy Sciences, Aname) for 1 h at 4 8C in darkness. Then, cells were rinsed in 0.1 M cacodylate buffer and bidistilled water, dehydrated through acetone series and embedded in Spurr’s
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resin (Agar Scientific LTD, Leica) according to standard protocols. Ultrathin sections (80 nm) were cut on an Ultramicrotome (LKB supernova, Leica) with a diamond knife, stained for 30 min with 2% uranyl acetate (Panreac Quı´mica S.A., Barcelona, Spain) and for 10 min with lead citrate (Electron Microscopy Sciences, Aname) and then examined in a transmission electron microscope (Zeiss EM-910) operating at 60–80 kV. 2.7. Statistical analysis All data are presented as the mean S:E:M. Analysis of variance (ANOVA) was made using the program SPSS 7.5 for Windows. The significance level considered was P < 0:05.
3. Results 3.1. Formation of cell cultures At the time of plating, many tissue fragments were suspended in the supernatant and cells appeared single or grouped. After 36 h, tubule fragments from caput and corpus epididymal segments began to attach to the bottom of the culture dishes. However, cauda epididymal fragments required 72 h to become firmly attached. Then, the cells began to migrate out of tubule fragments and acquired a flattened polygonal configuration with a patch-like growth pattern (Fig. 1A). During the subsequent culture period, the areas covered by epididymal cells became larger and after 12–16 days, a uniform confluent monolayer (80–90% of the floor of the well covered by cells) of tightly packed epithelioid cells was formed (Fig. 2A, C, and E). These monolayers were maintained for more than 60 days in culture. Samples from the three regions of epididymis showed the same pattern of development and no statistical differences in confluence of the monolayers were observed in any of the analyzed intervals. Some cells from the supernatant also adhered to the culture plate and formed colonies which were not associated to any fragment. These colonies grew and also contributed to the formation of the cell monolayer (Fig. 1B). Although cultures were usually initiated with 30 tubule fragments per well, an equivalent confluent monolayer of tightly packed epithelioid cells was formed after 12–16 days in wells seeded with 15, 60, or 90 fragments per well from the three regions of epididymis. These monolayers were also maintained for more than 60 days in culture and statistical differences in the confluence of the monolayers were not observed among the tested fragment densities. 3.2. Morphology and viability of cultured cells The cultured cells of caput, corpus, and cauda regions of boar epididymis displayed similar morphological characteristics. In the first week, the cells were flattened and polygonal and showed mutual contacts through cell bodies and cytoplasmic extensions, although intercellular spaces were present in the monolayers. They had a central nucleus, with one or two visible nucleoli, and contained numerous granules in the cytoplasm
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Fig. 1. Phase-contrast micrographs of epithelial cells from caput epididymis of Sus domesticus after 3 days in culture. (A) The tissue fragment (T) has plated out and epithelial cells have migrated and attached on the bottom of the culture well. (B) Cells from the supernatant have also adhered to the culture plate forming a colony. N, nuclei. Magnification: 350.
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Fig. 2. Phase-contrast micrographs of monolayers of (A) caput, (C) corpus, and (E) cauda epididymal epithelial cells (175) and immunostaining with anti-cytokeratin antibody of (B) caput, (D) corpus, and (F) cauda monolayers (100) of Sus domesticus after 14 days in culture.
(Fig. 1A and B). After 35 days in culture, the shape of the cultured cells was similar to that of the first week, except that they were more flattened and more tightly packed in the monolayer and contained numerous vesicles in their cytoplasm. These epididymal cells remained viable and maintained the morphological characteristics described earlier, for 2 months in culture. 3.3. Culture composition The percentages of cytokeratin-positive cells within the monolayers from caput, corpus, and cauda epididymis, at different days in culture, are shown in Fig. 3. All cultures showed about 80% of cytokeratin-positive cells for 49 days and no statistical differences were
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Fig. 3. Percentage of cytokeratin-positive cells from caput, corpus, and cauda cultures after different days in culture (n ¼ 3).
observed between caput, corpus, and cauda cultures. In one experiment, the percentages of cytokeratin-positive cells were also determined after 62 days, with 75.7, 87.9, and 86.0% in caput, corpus, and cauda cultures, respectively. These results indicate that most of the cells in the confluent monolayers were epithelial cells (Fig. 2B, D, and F). Non-epithelial cells were always found to be present in lower percentages and no cultures with overgrowth of non-epithelial cells were observed in this study. 3.4. Proliferation of epithelial epididymal cells in culture Mitotic figures were regularly observed in samples from caput, corpus, and cauda cultures that were double stained with bisbenzimide and anti-cytokeratin antibody. All nuclei observed in division belonged to cytokeratin-positive cells, indicating that proliferation of epithelial cells was occurring. Mitotic nuclei were not observed in cytokeratinnegative cells. In secondary cultures, epididymal cells rapidly attached to the substrate and formed confluent monolayers with densely packed polygonal cells similar to those of primary cultures. The number of cells in these secondary cultures doubled three days after trypsinization. Therefore, the percentage of cytokeratin-positive cells was comparable to that of the primary cultures. Overgrowth of non-epithelial cells was not observed. 3.5. Ultrastructure of cultured epididymal epithelial cells As assessed by transmission electron microscopy, the boar epididymal epithelial cells from caput, corpus and cauda cultures had similar ultrastructural characteristics throughout the 35 days of culture with no evidence of degenerative or de-differentiation changes. Boar epididymal epithelial cells from the three epididymal regions formed a monolayer on the polycarbonate insert and their basal membranes displayed cytoplasmic insertions projecting into the pores of the insert. The apical surface of these cells was in contact with the culture medium and presented short stereocilia (Fig. 4A–C). Epithelial cells were tightly adhered to each other through interdigitations, desmosomes and tight junctions situated on their lateral and basal membranes (Fig. 4A–C), though some intercellular spaces were apparent. In general, the nuclei were oval with prominent nucleoli. An extensive rough
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Fig. 4. Electron micrographs of epididymal epithelial cells of Sus domesticus from (A) caput, (B) corpus, and (C) cauda cultures after 14, 28, and 35 days, respectively. The cells show apical stereocilia (SC) and a central nucleus (N) with nucleolus (NU). The cytoplasm contains mitochondria (M), rough endoplasmic reticulum (RER) and Golgi apparatus (G). Small apical pinocytotic vesicles (VP) and bigger secretory vesicles (VG) are also present. There are also lipid droplets (LI) and dense granules (DG). Note the cells adhere tightly to each other by means of junctional complexes (arrows), though some intercellular space is also apparent. Magnification: 9450.
endoplasmic reticulum, numerous mitochondria and a well-developed Golgi apparatus were observed in the cytoplasm of cultured cells. Multivesicular bodies, residual bodies, lipid droplets, dense granules and bundles of 10 nm filaments were also present. Numerous small electron lucid vesicles were particularly concentrated in the apical cytoplasm of cultured cells, while bigger vesicles were found in a perinuclear location (Fig. 4A–C).
4. Discussion Functional epididymal culture systems have been successfully achieved for several species, such as rodent [18,19,24–27], bovine [28], and marsupial [16,29]. Similar results
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have also been obtained in humans [13,17,30]. However, as far as boars are concerned, only one report—on the efferent duct—is available [31]. The culture system for boar epididymal epithelial cells described in this study varies from the protocol for the boar efferent duct in that longer periods of enzymatic digestion are needed to prepare epididymal tubule fragments. This is probably due to the large amount of connective tissue surrounding the epididymal tubules. We have observed that the length of the digestion period is an important factor in the successful establishment of epididymal cell monolayers. Fragments from the caput and corpus epididymis need similar enzymatic digestion periods; however, fragments from the cauda region need longer periods of enzymatic digestion to form a functional monolayer. This may be explained by the fact that there is more connective tissue surrounding the boar epididymal duct in the cauda region [32]. In humans, the cauda region failed to produce functional cells because the basal lamina of the connective tissue and smooth muscle was so thick that it could not be adequately digested with collagenase [3,30,33]. Another factor that has to be taken into account when attempting to culture epididymal epithelial cells is the incubation temperature. It is known that in most mammals the scrotal temperature of epididymis is 3–4 8C lower than basal body temperature. For this reason, one might expect that epididymal cell cultures would function more optimally at 33–35 8C, rather than at 37–38 8C. In addition, some authors have shown that the temperature has a marked effect on protein secretion and the expression of specific mRNAs [19,25,37]. However, according to Moore [3], epididymal cell cultures maintain the same morphological characteristics at 37 8C as they do at 33 8C. Importantly, the survival of spermatozoa and the induction of their maturation after co-incubation with epididymal epithelial cells are promoted at 37–38 8C [13,14,16,28,30]. Some authors mention the importance of an optimal initial cellular concentration for successful epididymal cell cultures [18,31]. Raczek et al. [15] observed that when too few fragments were seeded, confluence was not reached. According to the same author, in the absence of absolute epithelial cell numbers, the choice of the appropriate fragment to seed was arrived at from experience and the appearance of the tissue after processing. We tested four different densities of tubule fragments per well and an equivalent confluent monolayer of tightly packed epithelioid cells was formed in all cases. From our experience, a density of 30 fragments per well was suitable for obtaining epididymal epithelial cell cultures from caput, corpus and cauda regions. The fragments from the three regions of boar epididymis showed different patterns of attachment. Fragments from the caput and corpus attached themselves to the bottom of the culture dishes after 36 h, whereas cauda fragments required 72 h to attach firmly. This may be due to the fact that, in boars, the epididymal tubules of the cauda are greater in diameter than those of the caput and corpus [32]. In contrast, epididymal fragments from bulls attached to the culture plate within the first 12 h [28], from rodents within 18 h [27], from marsupials within 24–48 h [16] and from humans within 3–6 days [17,30]. Boar epididymal cell monolayers from caput, corpus and cauda regions reach 80–90% confluence after 12–16 days in culture. The same confluence is reached after 3–7 days in marsupials [16] as well as in rodents [18,19,24–27], 5–7 days in bulls [28] and 8–13 days in humans [17,34]. The period that epididymal cells remained attached also differs among species. Boar epididymal cells remained attached for up to 2 months, whereas
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human cells detached after 6–8 weeks [17,30] and rat cells degenerated after 9–14 days [24,26,35]. Furthermore, we observed isolated epididymal epithelial cell colonies originating from single cells sloughed from tubule fragments by the digestion of collagenase. These colonies grow and form part of the cell monolayers in cultures from all three regions of the epididymis. In contrast, in marsupials, single epithelial cells showed no firm attachment to the floor of culture wells and degenerated [16]. Nevertheless, our results are not surprising, since some authors obtained the cell monolayers from a purified suspension of single principal cells [7–12]. Using whole epididymal tubule fragments produces primary cultures which contain epithelial and non-epithelial cells. In this study, we measured the percentage of epididymal epithelial cells assessed, as we mentioned earlier, by immunofluorescence of cytokeratins, which are recognized markers of epithelial cells [8,35]. About 80% of the cells were cytokeratin-positive in all caput, corpus and cauda cultures during the culture period. These results indicate that epithelial cells dominated in the monolayers. Moreover, the degree of epithelial cell enrichment obtained by the method described here is similar to that obtained after cell separation and centrifugation through a Percoll gradient [36] which is, however, a longer and more complex method. Only a small proportion of cells failed to react with the cytokeratin antibody and that proportion remained constant during the culture period. This shows that there was no massive overgrowth of non-epithelial cells in these cultures and is further evidence that epithelial cells actively inhibit the proliferation of non-epithelial cells, as has been described by Lin et al. [16] and Akhondi et al. [30]. Nevertheless, several authors suggest that the presence of some non-epithelial cells in epididymal cultures is actually beneficial. According to Lin et al. [29] factors from the peritubular cells are critical for the propagation and maintenance of epididymal epithelial cell layers under culture conditions. Carballada and Saling [19] suggest that fibroblast factors may act to refine the epithelial pattern by inducing tissue-specific protein secretion. Peritubular cells may also provide the epididymal epithelial cells with some protection from the impact of enzymatic digestion during culture preparation [16]. It is well established that cultured epididymal epithelial cells appear to grow from tubule fragments by migration. Some authors have demonstrated that they also grow by mitotic proliferation, using specific proliferation assays like 3 H-thymidine [7,11,19] and 5-bromo20 -deoxyuridine [37] labeling. In this study, we were able to corroborate these results because we regularly observed mitotic figures in cytokeratin-positive cells from caput, corpus and cauda cultures. As we observed in electron microscopy, cultured boar epididymal epithelial cells from caput, corpus, and cauda regions present similar ultrastructural characteristics with no signs of degeneration or de-differentiation, during the entire culture period. The morphology of epididymal epithelial cells change markedly in culture from a columnar or cuboidal shape to a flattened, polygonal shape. This results in the height of these cells being lower than they are in vivo and makes difficult to distinguish between the various types of epithelial cells. However, the ultrastructural study suggests that cell monolayers are basically formed by principal epithelial cells. The cultured cells had certain features that characterize the epididymal epithelium in situ. These features include cell polarity,
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epithelial integrity and absorptive and secretory activities. Cell polarity was evident since numerous stereocilia were frequently found on the apical surface of cultured cells. As was the case in other studies, the stereocilia of epididymal epithelial cells were shorter than they were in vivo [12,38,39]. Epithelial integrity was maintained by interdigitations and junctional complexes in lateral and basal membranes. Absorptive activity was suggested by the presence of numerous pinocytotic vesicles in the apical cytoplasm, multivesicular bodies and residual bodies. The presence of an extensive rough endoplasmic reticulum, a well-developed Golgi apparatus, plenty of ribosomes and some bigger vesicles in perinuclear location suggest that the boar epididymal cells in culture maintained their capacity to synthesize and segregate proteins. This is the first report describing a protocol to culture boar epididymal epithelial cells from the caput, corpus and cauda regions. This study shows that epididymal epithelial cells can be maintained in vitro for more than 2 months without losing the morphological characteristics of native cells and suggests that they also retain their functional characteristics in culture. Being able to culture different epididymal regions is potentially a valuable tool for studying epididymal secretions and for analyzing regional differences. This system may also be used for coculture with boar spermatozoa in order to study the mechanism of sperm maturation, which remains poorly understood.
Acknowledgements Technical help from the Microscopy Service of the University of Girona is highly appreciated. Grant support: Projects AGL 2002-01924 (GAN-ACU) of Ministerio de Ciencia y Tecnologı´a (MCYT), entitled ‘‘Development of a methodology for the in vitro maturation of spermatozoa from Pie´ train boar’’ and RZ02-013 of Ministerio de Ciencia y Tecnologı´a (MCYT) and Instituto Nacional de Investigaciones Agrarias (INIA), entitled ‘‘Improvement of sperm quality and fertility ability of immature sperm from Iberic boars after in vitro coculture with epididymal epithelial cells.’’
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[7] Kierszenbaum AL, Lea O, Petrusz P, French FS, Tres LL. Isolation, culture, and immunocytochemical characterization of epididymal epithelial cells from pubertal and adult rats. Proc Natl Acad Sci USA 1981;78:1675–9. [8] Olson GE, Jonas-Davies J, Hoffman LH, Orgebin-Crist MC. Structural characterization of isolated rat epididymal epithelial cells. Gamete Res 1982;6:161–78. [9] Klinefelter GR, Amann RP, Hammerstedt RH. Culture of principal cells from the rat caput epididymidis. Biol Reprod 1982;26:885–901. [10] Joshi MS. Isolation and cell culture of the epithelial cells of cauda epididymidis of the bull. Biol Reprod 1985;33:187–200. [11] Wagley LM, Versluis TD, Brown DV, Amann RP. Culture of principal cells from the ram epididymis. A comparison of the morphology of principal cells in culture and in situ. J Androl 1984;5:389–408. [12] Byers SW, Djakiew D, Dym M. Structural features of rat epididymal epithelial cells in vitro. J Reprod Fertil 1985;75:401–11. [13] Moore HD, Curry MR, Penfold LM, Pryor JP. The culture of human epididymal epithelium and in vitro maturation of epididymal spermatozoa. Fertil Steril 1992;58:776–83. [14] Bongso A, Trounson A. Evaluation of motility, freezing ability and embryonic development of murine epididymal sperm after coculture with epididymal epithelium. Hum Reprod 1996;11:1451–6. [15] Raczek S, Yeung CH, Wagenfeld A, Hertle L, Schulze H, Cooper TG. Epithelial monolayers from human epididymal and efferent duct tubules testosterone metabolism and effects of culture conditions on cell height and confluence. Epithelial Cell Biol 1994;3:126–36. [16] Lin M, Zhang X, Murdoch R, Aitken RJ. In vitro culture of brushtail possum (Trichosurus vulpecula) epididymal epithelium and induction of epididymal sperm maturation in co-culture. J Reprod Fertil 2000;119:1–14. [17] Cooper TG, Yeung CH, Meyer R, Schulze H. Maintenance of human epididymal epithelial cell function in monolayer culture. J Reprod Fertil 1990;90:81–91. [18] Byers SW, Citi S, Anderson JM, Hoxter B. Polarized functions and permeability properties of rat epididymal epithelial cells in vitro. J Reprod Fertil 1992;95:385–96. [19] Carballada R, Saling PM. Regulation of mouse epididymal epithelium in vitro by androgens. J Reprod Fertil 1997;110:171–81. [20] Yeung CH, Cooper TG, Meyer R. Immature rat epididymal epithelial cells grown in static primary monolayer culture on permeable supports. II. Histochemistry and ultrastructure. Cell Tissue Res 1989;256: 573–80. [21] Castellon EA, Balbontin JB. Secretion of glycosidases in human epididymal cell cultures. Arch Androl 2000;45:35–42. [22] Montiel EE, Huidobro CC, Castellon EA. Glutathione-related enzymes in cell cultures from different regions of human epididymis. Arch Androl 2003;49:95–105. [23] Smith CA, Hartman TD, Moore HD. A determinant of Mr 34,000 expressed by hamster epididymal epithelium binds specifically to spermatozoa in co-culture. J Reprod Fertil 1986;78:337–45. [24] Sun GH, Lin YC, Cha TL, Yu DS, Chang SY, Liu HW. Conjugation of maturation-related wheat-germlectin-binding proteins to caput epididymal sperm in co-cultures with corpus epididymal epithelial cells of BALB/c mouse. Arch Androl 2000;45:43–52. [25] Kirchhoff C, Carballada R, Harms B, Kascheike I. CD52 mRNA is modulated by androgens and temperature in epididymal cell cultures. Mol Reprod Dev 2000;56:26–33. [26] Cooper TG, Yeung CH, Meyer R. Immature rat epididymal epithelial cells grown in static primary monolayer culture on permeable supports. I. Vectorial secretion. Cell Tissue Res 1989;256:567–72. [27] Chen YC, Bunick D, Bahr JM, Klinefelter GR, Hess RA. Isolation and culture of epithelial cells from rat ductuli efferentes and initial segment epididymidis. Tissue Cell 1998;30:1–13. [28] Gagnon A, Sullivan R, Sirard MA. Epididymal epithelial cells cultured in vitro prolong the motility of bovine sperm. J Androl 2000;21:842–7. [29] Lin M, Hess R, Aitken RJ. Induction of sperm maturation in vitro in epididymal cell cultures of the tammar wallaby (Macropus eugenii): disruption of motility initiation and sperm morphogenesis by inhibition of actin polymerization. Reproduction 2002;124:107–17. [30] Akhondi MA, Chapple C, Moore HD. Prolonged survival of human spermatozoa when co-incubated with epididymal cell cultures. Hum Reprod 1997;12:514–22.
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[31] Heiniger BM, Stoffel MH, Friess AE. Ultrastructural validation of an improved culture system for boar efferent duct epithelium. J Reprod Fertil 1996;106:251–8. [32] Stoffel MH, Friess AE. Morphological characteristics of boar efferent ductules and epididymal duct. Microsc Res Tech 1994;29:411–31. [33] Moore HD. Contribution of epididymal factors to sperm maturation and storage. Andrologia 1998;30: 233–9. [34] Sun GH, Liu HW, Lin YC, Yu DS, Chang SY. Identification of maturation-related wheat-germ lectinbinding proteins in the culture of human corpus epididymal epithelial cells. Arch Androl 2000;45:53–60. [35] Olson GE, Jonas-Davies J, Hoffman LH, Orgebin-Crist MC. Structural features of cultured epithelial cells from the adult rat epididymis. J Androl 1983;4:347–60. [36] Finaz C, Boue F, Meduri G, Lefevre A. Characterization of rat epithelial epididymal cells purified on a discontinuous Percoll gradient. J Reprod Fertil 1991;91:617–25. [37] Pera I, Ivell R, Kirchhoff C. Body temperature (37 8C) specifically down-regulates the messenger ribonucleic acid for the major sperm surface antigen CD52 in epididymal cell culture. Endocrinology 1996;137:4451–9. [38] White MG, Huang YS, Tres LL, Kierszenbaum AL. Structural and functional aspects of cultured epididymal epithelial cells isolated from pubertal rats. J Reprod Fertil 1982;66:475–84. [39] Klinefelter GR, Hamilton DW. Organ culture of rat caput epididymal tubules in a perifusion chamber. J Androl 1984;5:243–58.
In vitro culture of epithelial cells from the caput, corpus, and cauda epididymis of Sus domesticus Judit Bassols*, Elisabeth Ka´da´r, M Dolors Briz, Elisabeth Pinart, Sı´lvia Sancho, Nu´ria Garcia-Gil, Elena Badia, Anna Pruneda, Eva Bussalleu, Marc Yeste, Sergi Bonet Biotechnology of Porcine Reproduction, Department of Biology, Faculty of Sciences, University of Girona, Campus Montilivi sn, 17071 Girona, Spain Received 6 August 2003; received in revised form 10 December 2003; accepted 14 December 2003
Abstract This work describes a protocol to culture epididymal epithelial cells from the caput, corpus, and cauda regions of Sus domesticus. Epididymal epithelial fragments were obtained by dissection and enzymatic digestion with collagenase. About 30 epididymal fragments from each epididymal region were cultured in 24-well culture plates with supplemented RPMI-1640 medium at 37 8C, 5% CO2 in air, and 100% humidity. A confluent monolayer of polygonal and tightly packed epithelioid cells from the three epididymal regions was obtained after 12–16 days in culture and maintained in vitro for more than 60 days. The proportion of epididymal epithelial cells in these cultures was assessed by immunofluorescent staining for cytokeratins. Throughout the 2 months of culture, about 80% of the cells were cytokeratin-positive. Electron microscopy observations indicated that cultured cells from caput, corpus, and cauda epididymal regions were tightly adhered to each other by junctional complexes and that stereocilia were present in their apical membranes. Moreover, the presence of an extensive rough endoplasmic reticulum, Golgi apparatus and numerous vesicles in the cytoplasm suggested that cultured cells maintained secretory and absorptive activities. These results show that the epididymal epithelial cells in culture from S. domesticus retain some fundamental features that characterize the epididymal epithelium in the intact organ. This system might be a valuable tool for studying the mechanism of sperm maturation in vitro, including epididymal cell secretions and the analysis of regional differences. # 2004 Elsevier Inc. All rights reserved. Keywords: Boar; Sus domesticus; Epididymis; Epithelium; Epithelial cells; In vitro culture; Ultrastructure
* Corresponding author. Tel.: þ34-972418366; fax: þ34-972418150. E-mail address: [email protected] (J. Bassols).
0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2003.12.015
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1. Introduction It has been demonstrated that, in mammals, the epididymis plays an essential role in sperm maturation and storage [1]. Sperm maturation, which takes place during the transit of spermatozoa through the different epididymal regions [2], is a critical process in the acquisition of sperm motility and fertilizing ability. Functional changes during this process are the result of interaction between spermatozoa and epididymal epithelial cells, which need to be mediated by sequential changes in the protein composition of the epididymal fluid [3]. Different methods has been used to purify and identify many epididymal secreted proteins, but their role and importance in sperm physiology still needs to be established more fully [4–6]. Moreover, most of these methods make it difficult to study the activities of the epididymal epithelium without interference of testicular and non-epithelial tissue factors. To overcome this limitation, primary cultures of epididymal epithelial cells have been devised in different animal models. These in vitro cultures provide an attractive and simplified model system for the analysis of epithelial cell metabolic activities and their regulation because the synthesis and secretion of specific components can be monitored under defined conditions. In addition, data related to sperm maturation are provided by coculture experiments where intimate interactions between spermatozoa and epididymal epithelial cells can be examined in detail [16,23,24]. Some of the protocols employed to culture epididymal epithelial cells involve the isolation of a purified suspension of single epithelial cells [7–12]. However, the disruption of the epithelial architecture and the lack of basal lamina and other peritubular elements compromise the cells, which rapidly de-differentiate and lose function in culture. Currently, most methods use fragments or plaques of epithelium prepared after enzyme digestion [13–16]. Although differences in the enzymatic digestion procedure and the culture medium used have been observed, these protocols can generate cell monolayers in which epididymal epithelial cells retain their morphological characteristics, remain polarized and maintain absorptive and secretory activities, mimicking in vivo conditions [17,18]. Different studies have demonstrated that cultured epididymal epithelial cells can metabolize testosterone to the active metabolite, dihydrotestosterone [15] and synthesize and secrete alkaline phosphatase, acid phosphatase and N-acetylglucosaminidase [17,20]. Other secretory proteins, such as glycosidases [21] and glutathione-related enzymes [22] have also been described in epididymal cell cultures. In addition, an increase in progressive sperm motility and enhanced sperm survival has been shown in coculture with epididymal epithelial cells [13,14]. Moreover, it has been observed that several maturation-related compounds expressed by epididymal epithelium bind to spermatozoa in co-culture [23,24]. The aim of this study was to develop a cell culture system capable of supporting epithelial cells from caput, corpus, and cauda epididymis of Sus domesticus. This system will be used to identify and characterize boar epithelial cell secretions in specific regions of the epididymis. It will also be used for coculture with immature spermatozoa in order to study the sperm maturation process and the specific interactions between spermatozoa and epididymal epithelial cells in boars.
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2. Materials and methods 2.1. Animals Epididymal tissue was obtained from Pietrain and Iberian boars of 8–12 months of age. Once the animals were slaughtered, the testis and epididymis were located, transferred to sterile PBS (Gibco, Invitrogen S.A., Barcelona, Spain), supplemented with 50 U ml1 penicillin G and 50 mg ml1 streptomycin (Gibco, Invitrogen S.A.) and brought to the laboratory within 45–60 min. The boars were slaughtered by a veterinarian following approved guidelines for the ethical treatment of animals. 2.2. Tissue isolation and cell culture The epididymidis were removed from the testis and separated into three different segments: caput, corpus and cauda. Each epididymal segment was placed in a 150 mm culture dish (Nunc, LabClinics, Barcelona, Spain), covered with supplemented PBS and minced into fragments (2–5 mm in length). After three washes with supplemented PBS, tubule fragments were transferred into supplemented PBS containing 300 U ml1 collagenase (type VII, Sigma, Madrid, Spain) and incubated at 37 8C in a water bath for 2 h (in the case of the caput and corpus fragments) or 3 h (in the case of the cauda fragments). After incubation, tubule fragments (settled by gravity) were washed in three changes of the culture medium (see below). The supernatant was discarded and the pellet was resuspended with culture medium containing 150 U ml1 collagenase. Tubule fragments of caput, corpus, and cauda regions were incubated at 37 8C in a water bath, for 1, 2, and 3 h, respectively. After washing in culture medium, three times for 5 min at 200 g, usually about 30 tubule fragments were transferred to each well of a 24-well culture plate (Nunc, LabClinics) in 1 ml fresh culture medium and incubated at 37 8C in 5% CO2 in air and 100% humidity. After the first 24 h and then every 48 h, about half of the medium was changed during the first six days of culture. From then on, 90% of the medium was replaced every 48 h. Culture samples prepared for electron microscopy were treated as described above, except that 15 tubule fragments were plated into culture plate polycarbonate inserts (12 mm in diameter, 0.4 mm pore size; Millipore Ibe´ rica S.A., Madrid, Spain) in 500 ml culture medium. The inserts were then placed in a 24-well culture plate in 1 ml fresh culture medium. For immunofluorescence, 30 tubule fragments were plated into 0.1% gelatin (Merck, Madrid, Spain) pre-coated cover slips into a 24-well culture plate in 1 ml fresh culture medium. 2.3. Culture medium preparation The medium used for boar epididymal cell cultures was RPMI medium (Gibco, Invitrogen S.A.) supplemented with 10% of fetal calf serum (FCS) (PAA Laboratories, LabClinics), 1 mM D-sodium pyruvate (Gibco, Invitrogen S.A.), 100 nM insulin (Sigma), 200 nM hydrocortisone (Sigma), 200 nM testosterone (Fluka, Madrid, Spain), 1 mM dihydrotestosterone (Fluka), 5 mg ml1 transferrin (Sigma), 1 mg ml1 retinol acetate
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(Sigma), 25 mM Hepes (Gibco, Invitrogen S.A.), 50 U ml1 of penicillin G and 50 mg ml1 streptomycin (Gibco, Invitrogen S.A.). The culture medium was filtered with 0.2 mm filters (Pall-Gelman Laboratory, An Arbor, USA) and stored at 4 8C. 2.4. Immunofluorescence The percentages of epididymal epithelial cells in culture were assessed by immunofluorescent localization using a cytokeratin monoclonal antibody. At 14, 21, 28, 35, 42, and 49 days in culture, cells growing on 0.1% gelatin pre-coated cover slips were fixed with 3% p-formaldehyde (Merck) and 60 mM saccharose (Merck) in 0.1 M PBS (Merck) for 30 min. After three 10-min washes with 10 mM PBS, the cells were permeabilized for 10 min in a solution of 10 mM PBS containing 0.1% Triton X-100 (Sigma). Then cells were rinsed in 10 mM PBS for 5 min and incubated for 10 min in a blocking solution consisting of 10 mM PBS with 20 mM glycine (Serva, Innogenetics, Gent, Belgium) and 1% BSA (Sigma). The cells were stained with a mouse anti-pan cytokeratin monoclonal antibody (Chemicon, Pacisa-Giralt, Barcelona, Spain) (1:20) for 45 min at 37 8C, rinsed twice in PBS for 10 min and incubated in a 1:50 dilution of the rabbit anti-mouse immunoglobulin antibody conjugate FITC (Dako Diagnosticos S.A., Barcelona, Spain) for 30 min at 37 8C. After two 10-min washes in PBS, the cells were stained with 5 mM bisbenzimide (Sigma) for 7 min to localize the nucleus. Finally, the cells on the cover slips were covered with mounting medium (Sigma) and examined under a Leica DMR-XA fluorescence microscope. The percentages of cytokeratin-positive cells were determined by flow cytometry (FACSCalibur, Becton Dickinson). In order to provide control samples for background fluorescence, cells were incubated with a primary irrelevant monoclonal antibody (Chemicon, Pacisa-Giralt). 2.5. Light microscopy Cells in the culture plates were examined every other day using an inverted phasecontrast microscope (Zeiss, Telaval 31) and photographs were taken through a Ricoh, XRX 3000 camera. The extent of the surface covered by cells (percentage of confluence) and morphological characteristics of cultured cells were performed on different days of culture. Cell concentration and viability was also determined using trypan blue exclusion test. 2.6. Electron microscopy Cells grown on polycarbonate cell culture inserts were recovered at 7, 14, 21, 28, and 35 days in culture and fixed with 2.5% glutaraldehyde (Electron Microscopy Sciences, Aname, Barcelona, Spain) in 0.1 M cacodylate buffer (Agar Scientific LTD, Leica, Barcelona, Spain) for 1 h at 4 8C. The cells were rinsed in 0.1 M cacodylate buffer and post-fixed in 0.1 M cacodylate buffer containing 0.1% OsO4 (Electron Microscopy Sciences, Aname) for 1 h at 4 8C in darkness. Then, cells were rinsed in 0.1 M cacodylate buffer and bidistilled water, dehydrated through acetone series and embedded in Spurr’s
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resin (Agar Scientific LTD, Leica) according to standard protocols. Ultrathin sections (80 nm) were cut on an Ultramicrotome (LKB supernova, Leica) with a diamond knife, stained for 30 min with 2% uranyl acetate (Panreac Quı´mica S.A., Barcelona, Spain) and for 10 min with lead citrate (Electron Microscopy Sciences, Aname) and then examined in a transmission electron microscope (Zeiss EM-910) operating at 60–80 kV. 2.7. Statistical analysis All data are presented as the mean S:E:M. Analysis of variance (ANOVA) was made using the program SPSS 7.5 for Windows. The significance level considered was P < 0:05.
3. Results 3.1. Formation of cell cultures At the time of plating, many tissue fragments were suspended in the supernatant and cells appeared single or grouped. After 36 h, tubule fragments from caput and corpus epididymal segments began to attach to the bottom of the culture dishes. However, cauda epididymal fragments required 72 h to become firmly attached. Then, the cells began to migrate out of tubule fragments and acquired a flattened polygonal configuration with a patch-like growth pattern (Fig. 1A). During the subsequent culture period, the areas covered by epididymal cells became larger and after 12–16 days, a uniform confluent monolayer (80–90% of the floor of the well covered by cells) of tightly packed epithelioid cells was formed (Fig. 2A, C, and E). These monolayers were maintained for more than 60 days in culture. Samples from the three regions of epididymis showed the same pattern of development and no statistical differences in confluence of the monolayers were observed in any of the analyzed intervals. Some cells from the supernatant also adhered to the culture plate and formed colonies which were not associated to any fragment. These colonies grew and also contributed to the formation of the cell monolayer (Fig. 1B). Although cultures were usually initiated with 30 tubule fragments per well, an equivalent confluent monolayer of tightly packed epithelioid cells was formed after 12–16 days in wells seeded with 15, 60, or 90 fragments per well from the three regions of epididymis. These monolayers were also maintained for more than 60 days in culture and statistical differences in the confluence of the monolayers were not observed among the tested fragment densities. 3.2. Morphology and viability of cultured cells The cultured cells of caput, corpus, and cauda regions of boar epididymis displayed similar morphological characteristics. In the first week, the cells were flattened and polygonal and showed mutual contacts through cell bodies and cytoplasmic extensions, although intercellular spaces were present in the monolayers. They had a central nucleus, with one or two visible nucleoli, and contained numerous granules in the cytoplasm
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Fig. 1. Phase-contrast micrographs of epithelial cells from caput epididymis of Sus domesticus after 3 days in culture. (A) The tissue fragment (T) has plated out and epithelial cells have migrated and attached on the bottom of the culture well. (B) Cells from the supernatant have also adhered to the culture plate forming a colony. N, nuclei. Magnification: 350.
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Fig. 2. Phase-contrast micrographs of monolayers of (A) caput, (C) corpus, and (E) cauda epididymal epithelial cells (175) and immunostaining with anti-cytokeratin antibody of (B) caput, (D) corpus, and (F) cauda monolayers (100) of Sus domesticus after 14 days in culture.
(Fig. 1A and B). After 35 days in culture, the shape of the cultured cells was similar to that of the first week, except that they were more flattened and more tightly packed in the monolayer and contained numerous vesicles in their cytoplasm. These epididymal cells remained viable and maintained the morphological characteristics described earlier, for 2 months in culture. 3.3. Culture composition The percentages of cytokeratin-positive cells within the monolayers from caput, corpus, and cauda epididymis, at different days in culture, are shown in Fig. 3. All cultures showed about 80% of cytokeratin-positive cells for 49 days and no statistical differences were
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Fig. 3. Percentage of cytokeratin-positive cells from caput, corpus, and cauda cultures after different days in culture (n ¼ 3).
observed between caput, corpus, and cauda cultures. In one experiment, the percentages of cytokeratin-positive cells were also determined after 62 days, with 75.7, 87.9, and 86.0% in caput, corpus, and cauda cultures, respectively. These results indicate that most of the cells in the confluent monolayers were epithelial cells (Fig. 2B, D, and F). Non-epithelial cells were always found to be present in lower percentages and no cultures with overgrowth of non-epithelial cells were observed in this study. 3.4. Proliferation of epithelial epididymal cells in culture Mitotic figures were regularly observed in samples from caput, corpus, and cauda cultures that were double stained with bisbenzimide and anti-cytokeratin antibody. All nuclei observed in division belonged to cytokeratin-positive cells, indicating that proliferation of epithelial cells was occurring. Mitotic nuclei were not observed in cytokeratinnegative cells. In secondary cultures, epididymal cells rapidly attached to the substrate and formed confluent monolayers with densely packed polygonal cells similar to those of primary cultures. The number of cells in these secondary cultures doubled three days after trypsinization. Therefore, the percentage of cytokeratin-positive cells was comparable to that of the primary cultures. Overgrowth of non-epithelial cells was not observed. 3.5. Ultrastructure of cultured epididymal epithelial cells As assessed by transmission electron microscopy, the boar epididymal epithelial cells from caput, corpus and cauda cultures had similar ultrastructural characteristics throughout the 35 days of culture with no evidence of degenerative or de-differentiation changes. Boar epididymal epithelial cells from the three epididymal regions formed a monolayer on the polycarbonate insert and their basal membranes displayed cytoplasmic insertions projecting into the pores of the insert. The apical surface of these cells was in contact with the culture medium and presented short stereocilia (Fig. 4A–C). Epithelial cells were tightly adhered to each other through interdigitations, desmosomes and tight junctions situated on their lateral and basal membranes (Fig. 4A–C), though some intercellular spaces were apparent. In general, the nuclei were oval with prominent nucleoli. An extensive rough
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Fig. 4. Electron micrographs of epididymal epithelial cells of Sus domesticus from (A) caput, (B) corpus, and (C) cauda cultures after 14, 28, and 35 days, respectively. The cells show apical stereocilia (SC) and a central nucleus (N) with nucleolus (NU). The cytoplasm contains mitochondria (M), rough endoplasmic reticulum (RER) and Golgi apparatus (G). Small apical pinocytotic vesicles (VP) and bigger secretory vesicles (VG) are also present. There are also lipid droplets (LI) and dense granules (DG). Note the cells adhere tightly to each other by means of junctional complexes (arrows), though some intercellular space is also apparent. Magnification: 9450.
endoplasmic reticulum, numerous mitochondria and a well-developed Golgi apparatus were observed in the cytoplasm of cultured cells. Multivesicular bodies, residual bodies, lipid droplets, dense granules and bundles of 10 nm filaments were also present. Numerous small electron lucid vesicles were particularly concentrated in the apical cytoplasm of cultured cells, while bigger vesicles were found in a perinuclear location (Fig. 4A–C).
4. Discussion Functional epididymal culture systems have been successfully achieved for several species, such as rodent [18,19,24–27], bovine [28], and marsupial [16,29]. Similar results
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have also been obtained in humans [13,17,30]. However, as far as boars are concerned, only one report—on the efferent duct—is available [31]. The culture system for boar epididymal epithelial cells described in this study varies from the protocol for the boar efferent duct in that longer periods of enzymatic digestion are needed to prepare epididymal tubule fragments. This is probably due to the large amount of connective tissue surrounding the epididymal tubules. We have observed that the length of the digestion period is an important factor in the successful establishment of epididymal cell monolayers. Fragments from the caput and corpus epididymis need similar enzymatic digestion periods; however, fragments from the cauda region need longer periods of enzymatic digestion to form a functional monolayer. This may be explained by the fact that there is more connective tissue surrounding the boar epididymal duct in the cauda region [32]. In humans, the cauda region failed to produce functional cells because the basal lamina of the connective tissue and smooth muscle was so thick that it could not be adequately digested with collagenase [3,30,33]. Another factor that has to be taken into account when attempting to culture epididymal epithelial cells is the incubation temperature. It is known that in most mammals the scrotal temperature of epididymis is 3–4 8C lower than basal body temperature. For this reason, one might expect that epididymal cell cultures would function more optimally at 33–35 8C, rather than at 37–38 8C. In addition, some authors have shown that the temperature has a marked effect on protein secretion and the expression of specific mRNAs [19,25,37]. However, according to Moore [3], epididymal cell cultures maintain the same morphological characteristics at 37 8C as they do at 33 8C. Importantly, the survival of spermatozoa and the induction of their maturation after co-incubation with epididymal epithelial cells are promoted at 37–38 8C [13,14,16,28,30]. Some authors mention the importance of an optimal initial cellular concentration for successful epididymal cell cultures [18,31]. Raczek et al. [15] observed that when too few fragments were seeded, confluence was not reached. According to the same author, in the absence of absolute epithelial cell numbers, the choice of the appropriate fragment to seed was arrived at from experience and the appearance of the tissue after processing. We tested four different densities of tubule fragments per well and an equivalent confluent monolayer of tightly packed epithelioid cells was formed in all cases. From our experience, a density of 30 fragments per well was suitable for obtaining epididymal epithelial cell cultures from caput, corpus and cauda regions. The fragments from the three regions of boar epididymis showed different patterns of attachment. Fragments from the caput and corpus attached themselves to the bottom of the culture dishes after 36 h, whereas cauda fragments required 72 h to attach firmly. This may be due to the fact that, in boars, the epididymal tubules of the cauda are greater in diameter than those of the caput and corpus [32]. In contrast, epididymal fragments from bulls attached to the culture plate within the first 12 h [28], from rodents within 18 h [27], from marsupials within 24–48 h [16] and from humans within 3–6 days [17,30]. Boar epididymal cell monolayers from caput, corpus and cauda regions reach 80–90% confluence after 12–16 days in culture. The same confluence is reached after 3–7 days in marsupials [16] as well as in rodents [18,19,24–27], 5–7 days in bulls [28] and 8–13 days in humans [17,34]. The period that epididymal cells remained attached also differs among species. Boar epididymal cells remained attached for up to 2 months, whereas
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human cells detached after 6–8 weeks [17,30] and rat cells degenerated after 9–14 days [24,26,35]. Furthermore, we observed isolated epididymal epithelial cell colonies originating from single cells sloughed from tubule fragments by the digestion of collagenase. These colonies grow and form part of the cell monolayers in cultures from all three regions of the epididymis. In contrast, in marsupials, single epithelial cells showed no firm attachment to the floor of culture wells and degenerated [16]. Nevertheless, our results are not surprising, since some authors obtained the cell monolayers from a purified suspension of single principal cells [7–12]. Using whole epididymal tubule fragments produces primary cultures which contain epithelial and non-epithelial cells. In this study, we measured the percentage of epididymal epithelial cells assessed, as we mentioned earlier, by immunofluorescence of cytokeratins, which are recognized markers of epithelial cells [8,35]. About 80% of the cells were cytokeratin-positive in all caput, corpus and cauda cultures during the culture period. These results indicate that epithelial cells dominated in the monolayers. Moreover, the degree of epithelial cell enrichment obtained by the method described here is similar to that obtained after cell separation and centrifugation through a Percoll gradient [36] which is, however, a longer and more complex method. Only a small proportion of cells failed to react with the cytokeratin antibody and that proportion remained constant during the culture period. This shows that there was no massive overgrowth of non-epithelial cells in these cultures and is further evidence that epithelial cells actively inhibit the proliferation of non-epithelial cells, as has been described by Lin et al. [16] and Akhondi et al. [30]. Nevertheless, several authors suggest that the presence of some non-epithelial cells in epididymal cultures is actually beneficial. According to Lin et al. [29] factors from the peritubular cells are critical for the propagation and maintenance of epididymal epithelial cell layers under culture conditions. Carballada and Saling [19] suggest that fibroblast factors may act to refine the epithelial pattern by inducing tissue-specific protein secretion. Peritubular cells may also provide the epididymal epithelial cells with some protection from the impact of enzymatic digestion during culture preparation [16]. It is well established that cultured epididymal epithelial cells appear to grow from tubule fragments by migration. Some authors have demonstrated that they also grow by mitotic proliferation, using specific proliferation assays like 3 H-thymidine [7,11,19] and 5-bromo20 -deoxyuridine [37] labeling. In this study, we were able to corroborate these results because we regularly observed mitotic figures in cytokeratin-positive cells from caput, corpus and cauda cultures. As we observed in electron microscopy, cultured boar epididymal epithelial cells from caput, corpus, and cauda regions present similar ultrastructural characteristics with no signs of degeneration or de-differentiation, during the entire culture period. The morphology of epididymal epithelial cells change markedly in culture from a columnar or cuboidal shape to a flattened, polygonal shape. This results in the height of these cells being lower than they are in vivo and makes difficult to distinguish between the various types of epithelial cells. However, the ultrastructural study suggests that cell monolayers are basically formed by principal epithelial cells. The cultured cells had certain features that characterize the epididymal epithelium in situ. These features include cell polarity,
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epithelial integrity and absorptive and secretory activities. Cell polarity was evident since numerous stereocilia were frequently found on the apical surface of cultured cells. As was the case in other studies, the stereocilia of epididymal epithelial cells were shorter than they were in vivo [12,38,39]. Epithelial integrity was maintained by interdigitations and junctional complexes in lateral and basal membranes. Absorptive activity was suggested by the presence of numerous pinocytotic vesicles in the apical cytoplasm, multivesicular bodies and residual bodies. The presence of an extensive rough endoplasmic reticulum, a well-developed Golgi apparatus, plenty of ribosomes and some bigger vesicles in perinuclear location suggest that the boar epididymal cells in culture maintained their capacity to synthesize and segregate proteins. This is the first report describing a protocol to culture boar epididymal epithelial cells from the caput, corpus and cauda regions. This study shows that epididymal epithelial cells can be maintained in vitro for more than 2 months without losing the morphological characteristics of native cells and suggests that they also retain their functional characteristics in culture. Being able to culture different epididymal regions is potentially a valuable tool for studying epididymal secretions and for analyzing regional differences. This system may also be used for coculture with boar spermatozoa in order to study the mechanism of sperm maturation, which remains poorly understood.
Acknowledgements Technical help from the Microscopy Service of the University of Girona is highly appreciated. Grant support: Projects AGL 2002-01924 (GAN-ACU) of Ministerio de Ciencia y Tecnologı´a (MCYT), entitled ‘‘Development of a methodology for the in vitro maturation of spermatozoa from Pie´ train boar’’ and RZ02-013 of Ministerio de Ciencia y Tecnologı´a (MCYT) and Instituto Nacional de Investigaciones Agrarias (INIA), entitled ‘‘Improvement of sperm quality and fertility ability of immature sperm from Iberic boars after in vitro coculture with epididymal epithelial cells.’’
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