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Isolation and culture of rat seminal vesicle epithelial cells
Experimental
Isolation
Copyright @ 1983 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827183/060293-12$02.00/0
Cell Research 145 (1983) 293-304
and Culture
The Use of the Secretory
of Rat Seminal Protein
ABRAHAM L. KIERSZENBAUM, and IKUMASA TAKENAKA
Vesicle
Epithelial
SVS IV as a Functional
ROBERT
M. DePHILIP,
Cells
Probe
W. AUSTIN
SPRUILL
Department of Anatomy and The Laboratories for Reproductive Biology, School of Medicine, University of North Carolina, Chapel Hill, NC 27514, USA
SUMMARY A method for the isolation and culture of seminal vesicle epithelial cells obtained from control and androgen-primed sexually-immature, uncastrated rats is described. This method allows the establishment of monolayer cultures from aggregates of seminal vesicle epithelial cells isolated after trypsin and collagenase digestion. Phase contrast and transmission electron microscopic methods demonstrate that cell aggregates, after attaching to the substrate, establish within 48 h a colony-like, epithelial-like growth pattern. Immunofluorescent localization studies of SVS IV, an androgen-dependent secretory protein purified from rat seminal vesicle secretion, show that cultured seminal vesicle epithelial cells are immunoreactive. An electrophoretic analysis of [35S]methionine-labeled secretory proteins immunoprecipitated with rabbit anti-SVS IV serum demonstrate that, whereas SVS IV is newly-synthesized and accumulated in the medium of cultured seminal vesicle cells established from androgen primed rats, cultured cells from control rats appear to synthesize and accumulate SVS IV in a precursor form. Results of this work show that seminal vesicle epithelial cells in culture not only retain several structural features representative of the tissue but also serve as a potential system for the study of androgen action.
The seminal vesicle is an androgen-dependent accessory gland of the male reproductive tract. Under the influence of androgens, the seminal vesicle develops and maintains a secretory function. After castration, the organ undergoes involution (for a review see [l]) accompanied by a decline in its remarkable protein and secretory activity [2-4]. Secretory products of the rat seminal vesicle [5], including several proteins [2, 6-81, accumulate in the glandular lumen and are discharged into the urethra at the time of ejaculation. In rodents, one of the functional properties of seminal vesicle secretion is to contribute to clot formation of the ejaculated semen [9]. Although the direct functional role of seminal vesicle secretion on sperm fertility has not been established, it has been recently reported that a protein secreted by rat seminal vesicles binds to ejacuiated sperm
UOI. Major effort has been devoted to the characterization of rat seminal vesicle secretory proteins and their differential regulation by androgens [7, 8, 11, 121. It has been proposed that androgens control the synthesis and secretion of rat seminal vesicle proteins by regulating changes in the steady-state pool size for specific mRNA levels [4, 121. Ostrowski et al. [71 have reported five major rat seminal vesicle secretory proteins designated SVS I to V according to their increasing electrophoretic mobility. One of these proteins, SVS IV (corresponding to protein S of Higgins et al. [2]), has been regarded as a potential marker for androgen action [4, 71 and for assessment of the functional development of rat seminal vesicle [ 131. 20-838331
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The value of isolated epithelial fragments of seminal vesicles [14] or isolated epithelial cells of seminal vesicles maintained for short-term incubation experiments [15] has been reported. An alternative approach for long-term incubation experiments is the use of primary cell cultures of seminal vesicle epithelial cells. Primary cell cultures yield homogeneous populations of cells able to maintain, for a period of time, a degree of functional differentiation in a well-controlled environment. In this paper, we report a simple method for the isolation and culture of seminal vesicle epithelial cells from sexually-immature rats. The functional status of cultured seminal vesicle epithelial cells was evaluated by both immunocytochemistry and immunoprecipitation of [35S]methionine-labeled secretory proteins using a specific rabbit serum generated against SW IV. The combined use of these immunological approaches suggests a differential effect on SVS IV gene expression in cultured epithelial cells established from seminal vesicles of control and androgen-primed rats.
MATERIALS Isolation
AND
and culture
METHODS of rat seminal
vesicle epithelial
cells
Primary cultures of rat seminal vesicle epithelial cells were established from sexually-immature rats (Charles River CD) according to the procedure outlined below. The rats from which cultures were prepared varied from 28 to 35 days, the exact ages being included in the description of results. Seminal vesicles were removed under sterile conditions and coagulating glands were carefully removed under a dissecting microscope. The glands were minced with scissors into the smallest pieces possible and the luminal secretion washed out in Hanks Balanced Salt Solution (HBSS), pH 7.6. Tissue minces were dissociated with 0.25 % trypsin (Sigma T-8128) in HBSS (5 mg of tissue per ml solution) at 32°C for 30-40 min with continuous agitation in a thermo-bath shaker (60 cycles/min) in an Brlenmeyer flask. The sample was then allowed to settle for 5 min. The supernatant was discarded and the sediment suspended in a solution of collagenase (Sigma C-0130) 1 mg/ml in HBSS and incubated at 32°C for 90 min in a shaker as above. The sample was allowed to stand at room temperature for 5 min, the supematant discarded and the resulting sediment was resuspended in Eagle’s Minimum Essential Medium supplemented with 10% fetal bovine serum (Sterile Systems, Inc.), 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 4 mM glutamine, 100 U/ml penicillin and 100 ug/ml streptomycin. The sample, consisting of clusters of epithelial cells and single cells, was plated in tissue culture flasks, dishes or on glass and plastic coverslips (approximate cell density: 1 x lO?ml) and incubated in a humidified COz-air incubator at 32°C. Media were changed every 24 h. To minimize the possible contamination with non-epithelial cells (mainly smooth muscle cells and tibroblasts), the plated sample was allowed to settle in a tissue culture flask for about l-4 h. Under these conditions, non-epithelial cells attach very rapidly to the substrate while aggregates of seminal vesicle epithelial cells remain in suspension. The suspension of cell aggregates is then transferred to a new culture flask or dish where most of them attach within 24 h and establish an epithelial-like, colony-like growth pattern. Contamination with non-epithelial cells determined by phase contrast microscopy and transmission electron microscopy was less than 5 %. TO study the induction of SVS IV in vivo, sexually-immature male rats (21 or 28 days old) were given daily subcutaneous injections of 0.1 mg testosterone propionate in 0.1 ml sesame oil. Control rats were injected with sesame oil alone. After one week of treatment, where rats were either 28 or 35 days old, seminal vesicles were isolated for epithelial cell culture. Post-trypsin and post-collagenase samples were processed for transmission electron microscopy to monitor the enzymatic digestion of tissue (see below).
Transmission electron microscopy (TEM) Seminal vesicle epithelial cells cultured on plastic coverslips (Thermanox) were rinsed in phosphatebuffered saline (PBS) and fixed in 2.5 % glutaraldehyde in 0.1 M phosphate buffer (pH 6.9) for 1 h at room temperature and post-fixed in 1% osmium tetroxide in the same buffer. Specimens were embedded in Maraglas (Polysciences) according to standard procedure. Blocks were sectioned with
Res
145 (1983
Secretion of SW IV in seminal
EXD Cell Res 145 (1983)
vesicle cell cultures
orientations either parallel or perpendicular to the substrate. Post-trypsin and post-collagenase samples were fixed and embedded as described above. One urn thick sections were stained with toluidine blue. Thin sections (gray-silver transmitted interference color) were collected on Formvarcoated copper electron microscope grids and stained first with many1 acetate and then with lead citrate. Thin sections were examined with a JEM 1OOB transmission electron microscope.
Indirect
immunoj7uorescence
Seminal vesicle epithelial cells grown on glass coverslips and maintained in serum-supplemented Eagle’s Minimum Essential Medium were rinsed in PBS, fixed with 3.7 % formaldehyde for 15 min and permeabilized with 0.2% Triton X-100 in PBS for 2 min. Cells were incubated in 1.5% normal goat serum before and after incubation with rabbit anti-SVS IV serum for 1 h in a moist chamber at room temperature (working dilutions: 1: 25, 1 : 50 and 1 : 75). To localize bound SVS IV antibody, fluorescein isothiocyanate-conjugated goat anti-rabbit IgG was applied to coverslips for 30 min. Coverslips were then rinsed in PBS and mounted with Elvanol [16]. Immunocytochemical controls included: (1) omission of the primary antiserum; (ii) absorption of anti-SVS IV with immunoafftnity purified SVS IV [7] carried out as described [17, 181; (iii) immunoprecipitation of [35S]methioninelabeled seminal vesicle secretory proteins (see below); and (iu) immunohistochemical localization of SVS IV in the epithelium of intact seminal vesicles (not shown).
[“Slmethionine-labeling of rat seminal vesicle
of cultured epithelial
cells
Primary cell cultures plated in 25 cm* tissue culture flasks were incubated with [“Slmethionine (sp. act. 1 100 Cilmmol, New England Nuclear) for 24 h between the 2nd and 3rd day after plating. Just before labeling, cultures were rinsed two times with serum-free, hormone-supplemented medium. This medium consisted of Eagle’s Minimum Essential Medium containing l/10 the usual concentration of methionine and supplemented with the following to the final concentration indicated: nonessential amino acids, 0.1 mM; glutamine, 4 mM; sodium pyruvate, 1 mM; penicillin, 100 U/ml; streptomycin, 100 @ml; insulin, 5 &ml; transferrin, 5 &ml; epidermal growth factor, 3 &ml; human growth hormone, 6.5 @J/ml and retinol, 10 PM. Dihydrotestosterone (0.1 uM) was included in the medium of cells cultured from androgen-primed seminal vesicles. Each group of cells (control and medium containing 100 androgen-primed) was incubated in serum-free, hormone-supplemented t&i/ml [35S]methionine and, at the end of the labeling period, the media were collected and phenylmethylsulfonyl fluoride added to a final concentration of 10 mM. The media were centrifuged (13000 g) 15 min, 4°C and the supernatant frozen (-20°C). Incubation was carried out at 32°C in a humidified atmosphere containing 5 % CO*.
Immunoprecipitation
of [35S]methionine-labeled
SVS IV
A volume of medium from cell cultures containing 500000 trichloroacetic acid (TCA)-precipitable cpm was taken for specific immunoprecipitation. One-fifth this volume of 5x immune buffer (5x; 250 mM NaCl, 2.5 % sodium deoxycolate, 2.5 % Nonidet P-40, 2.5 % methionine and 100 mM Tris-Cl, pH 7.5) was added and the volume brought to 600 pl with lx immune buffer. Samples were preabsorbed with 100 ul of a 10% w/v suspension of protein A bearing Sruph. aureus cells (Pansobin, Calbiochem) for 30 min at 23°C. The Pansorbin was removed by centrifugation (5 000 g, 10 min, 4°C) and 30 ul of a 1 : 50 dilution of SW IV specific rabbit antiserum was added. Purified SVS IV (25 ug) was added to indicated samples to test the specificity of immunoprecipitation. Samples were incubated for 1 h at 37°C and then for 2 days at 4°C. Samples were brought to room temperature, 100 pl of Pansorbin added and incubation continued for 30 min at 23°C. Finally, the mixture was layered over a 1 ml cushion of immune buffer containing 1 M sucrose and the immunoprecipitate collected by centrifugation (5 000 g, 20 min, 4°C). The pellet was resuspended and washed two times with lx immune buffer. The final pellet was resuspended in 100 pl of electrophoresis sample buffer (10% w/v glycerol, 5 % v/v 2-mercaptoethanol, 2.3 % w/v sodium dodecyl sulfate (SDS) and 0.0625 M Tris-Cl, pH 6.8) [19] and heated at 100°C for 10 min. After cooling to room temperature, samples were clarified (13 000 g, 10 min, 23°C) and the supematant fractionated by sodium dodecyl sulfate-poiyacrylamide gel electrophoresis (SDS-PAGE) [19] on a 14x 14 cmx 1.5 mm slab gel of 15 % acrylamide overlaid with a 4 cm stacking gel of 4.75% acrylamide. After electrophoresis for 14 h at 100 V, gels were stained with Coomassie blue R and processed for fluorography using EN3HANCE (New England Nuclear) according to the procedure of the manufacturer.
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Exp Cell Res 145 (1983)
Fig. 1. Trypsin-collagenase dissociated seminal vesicle fragments of a 30-day old rat. The epithelium consists of simple columnar cells. The large cytoplasmic vacuoles are probably a consequence of tissue manipulation. Remnant of seminal vesicle secretion (SW) is present at the luminal epithelial side of the cell aggregate. Arrow indicates a contaminating non-epithelial cell. Inset. Enzymaticallydissociated seminal vesicle epithelium (as above) showing a mitotic cell (metaphase) with vesicles containing secretory granules (arrowheads). Arrow points to microvilli. CC, Golgi complex region. x 1920; (inset) x2 230.
Exp Cell Res 145 (1983)
Secretion of SVS IV in seminal vesicle cell cultures
RESULTS Microscopic evaluation of enzymatically-dissociated vesicle epithelium
rat seminal
TEM studies of rat seminal vesicle tissue minces following trypsin and collagenase digestion demonstrate that most of the non-epithelial cell components of the stroma (mainly smooth muscle cells and tibroblasts) are removed (figs 1, 2) and that the samples, consisting of a predominant population of epithelial cells, retain the characteristic structural features reported in intact seminal vesicles [l]. Mitotic cells are occasionally observed (fig. 1). Seminal vesicle epithelial cells display a smooth basal surface which contrasts with the free apical region of the cell showing abundant microvillar extensions (fig. 2). Remnants of seminal vesicle secretion can be noted adjacent to the free apical cell region (fig. 1); Junctional complexes located at the apical, juxtaluminal cell region maintain the stability of epithelial cell clusters (fig. 2), a feature which fosters the establishment of a colony-like growth pattern after attachment of cell aggregates to the culture substrate (figs 3, 4). The characteristic topographic location of rough endoplasmic reticulum cisternae, Golgi complex regions and secretory vesicles, some of them displaying an eccentric electron-dense granule, are some of the most relevant cytoplasmic features observed in the seminal vesicle epithelium of control rats following enzymatic treatment (fig. 2). Similar features were observed in specimens from androgen-primed rats with the exception that the number of secretory vesicles was more abundant and the columnar epithelial cells taller (not shown). The cytological effects of testosterone propionate on the epithelium of rat seminal vesicles have been reported [20]. Morphological and immunocytochemical vesicle epithelial cells
evaluation of cultured rat seminal
After selective cell attachment of non-epithelial cells and re-plating of epithelial cell aggregates, as described in Materials and Methods, seminal vesicle epithelial cell aggregates from control and androgen-primed rats attach to the substrate within 24 h and establish an epithelial-like, colony-like growth pattern that is clearly apparent 48 h after plating (figs 3, 4). Primary cultures of seminal vesicle epithelial cells attain a confluent monolayer 5-7 days after plating. The size of the colony-like formations originating from cell aggregates of androgen-primed rats appear larger than those established from control rats (not shown). Because testosterone is known to induce in seminal vesicles of non-castrated rodents increased DNA synthesis accompanied by enhanced organ weight and DNA content in vivo [21], additional quantitative studies are required to determine in these cultures precise differences in cell number and volume, DNA synthetic activity and mitotic index between control and androgen-primed groups. Fig. 2. Detail of fig. 1 illustrating concentric arrays of rough endoplasmic reticulum cistemae (RER) at the basal cell region and the supranuclear location of Golgi complex saccules (CC) and secretory granules (arrozuheads). Arrows indicate linear densities corresponding to tight junctions between adjacent cells. MV, microvilli. x6 720.
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3. Phase contrast micrograph of seminal vesicle epithelial cells in a colony-like arrangement. indicates the central location of the originally-plated cell aggregate. 48 h after plating; 33-day old rat. x 330. Fig. 4. High magnification view of a colony-like formation of seminal vesicle epithelial cells displaying a characteristic epithelial-like organization. Phase contrast microscopy. 48 h after plating; 33-day old rat. X620. Fig. Arrow
Exp Cell Res 145 (1983)
Secretion of SVS IV in seminal
vesicle cell cultures
We wanted to determine general morphological features of cultured seminal vesicle epithelial cells after their adaptation to culture conditions. Primary cultures of control rats were studied by transmission electron microscopy 4 days after plating. Thin sections slightly oblique to the culture substrate (fig. 5) demonstrate that, although cultured seminal vesicle epithelial cells still retain numerous microvilli on the free apical cell surface (compare with figs 1 (inset), and 2), there is a considerable reduction in the size of secretory vesicles. Secretory vesicles located close to the microvillar cell region display an electrondense content (fig. 5). In general, the appearance, number and size of secretory vesicles in cultured cells differ from features observed in trypsin-collagenase tissue (fig. 2). An additional relevant feature observed in cultured seminal vesicle epithelial cells is the presence of tight junctions and desmosomes between adjacent cells (fig. 5). These junctional structures are regarded as indicative of epithelial cell nature [22]. As a result from the morphological analysis, it was assumed that, whereas seminal vesicle epithelial cells in culture lose some of the structural correlates of the intact seminal vesicle epithelium, they still maintained a certain degree of morphological differentiation. The next step in our study was the analysis of SVS IV immunoreactivity in cultured seminal vesicle epithelial cells established from control and androgenprimed rats using a specific rabbit anti-SVS IV serum. This study was carried out to further validate the epithelial nature of seminal vesicle cells in culture using a specific protein marker. A variable proportion of cultured seminal vesicle cells from 35day-old rats exhibited SVS IV immunoreactivity at the perinuclear region and scattered throughout the cytoplasm in the form of fluorescent spots (fig. 6A). This result is consistent with the fact that SVS IV is known to be detectable in sexually-immature animals of this age [13]. Under the same plating and culture conditions, almost all seminal vesicle cells cultured from androgenprimed rats displayed SVS IV immunoreactivity (fig. 6B-D). The specificity of SVS IV immunofluorescence was described in Materials and Methods. Synthesis of SVS IV in vitro We utilized SDS-PAGE to provide direct evidence that SVS IV is newly-synthesized by cultured seminal vesicle epithelial cells. Seminal vesicle secretory proteins from 28-day old rats that had received daily injections of testosterone propionate for 1 week were collected and fractionated by SDS-PAGE. Fig. 7 (lane I, Coomassie blue staining) shows that all the five major seminal vesicle secretory proteins [7] can be resolved. SVS IV is identified by its co-migration with purified SVS IV (fig. 7, lane 2, Coomassie blue staining). The seminal vesicles of control 28-day old rats, known to just begin to accumulate their secretory proteins [ 131, did not yield sufficient secretory product for electrophoretic analysis. Rabbit antibody specific for rat SVS IV was used to immunoprecipiFig. 5. TEM of cell components of a colony-like formation similar to fig. 4 (3 days after plating). A tight junction (arrou) and desmosomes (between crossed arrows) can be identified between adjoining cultured cells. The arrowheads indicate secretory vesicles near the microvillar (MV) cell region. CC, Golgi complex region; N, nucleus. x 14 400.
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6. Immunofluorescent localization of SVS IV in cultured rat seminal vesicle epithelial cells from control (35 days old); (B-D) androgen-primed (35 days old) rats. SVS IV immunoreactivity is more conspicuous in seminal vesicle cell cultures established from androgen primed rats. (D) Immunofluorescent granules which may correspond to the secretory granules depicted in TEMs (compare with fig. 5). The perinuclear immunofluorescence in the form of half-moon shaped patches (C) coexist with non-immunoreactive small vacoules. Dilution of anti-SVS IV serum: 1 : 75. (A, B) x370; (C) x375; (0) x 1280. Fig. (A)
tate [35S]methionine-labeled protein from the medium of epithelial cells derived from control and androgen-primed seminal vesicles (fig. 7, lanes 3 and 4, autoradiogram). We have found in medium from seminal vesicle epithelial cells cultured from control rats a very faint protein band co-migrating with SVS IV and a more intense protein band migrating more slowly than SVS IV (fig. 7, lane 3). Evidence that this higher MW band is antigenically related to SVS IV was provided by the ability of purified SVS IV to reduce the amount of radioactivity in this precipitated band (fig. 8, lane 2). A prominent [35S]methionine-labeled immunoprecipitated protein co-migrating with purified SVS IV was found in the medium of cultured seminal vesicle epithelial cells from androgen-primed rats (fig. 7, lane 4).
Exp Cell Res 145 (1983)
Secretion
of SW IV in seminal
vesicle cell cultures
67
Fig. 7. SDS-PAGE of seminal vesicle secretory proteins and SVS IV-specific immunoprecipitates from media of cells in culture. Secretory proteins in the seminal vesicle of a 28-day old, androgenprimed rat (lane I), purified SVS IV (lane 2) and [3’S]methionine-labeled, SVS IV-specific immunoprecipitates (lanes 3 and 4) were fractionated by SDS-PAGE on gels of 15 % a&amide following the procedure of Laemmli [ 191. The unlabeled secretory proteins (100 ug, lane 1) and purified SVS IV (10 pg. lane 2) were detected by Coomassie blue staining. [35S]Methionine incubation and immunoprecipitation were carried out as described in Materials and Methods. Equal amounts of radioactivity (5x 16 cpm) were added to the immunoprecipitation reaction. [35S]Methionine-containing proteins were detected by fluorography. 3, 4, Fluorographs of SVS IV-specific immunoprecipitates in the media of cells cultured from the tissue of 3, control; 4, androgen-primed rats. The five major seminal vesicle secretory proteins [7] are identified by Roman numerals and arrowheads. The positions of standard MW markers in kD are indicated on the right. Minor [35S]methionine-labeled protein bands in the immunoprecipitates presumably represent non-specific adsorption. Fig. 8. SVS IV-specific immunoprecipitation of [35S]methionine-labeled protein from the culture medium of cells from 28-day-old, untreated rats. Equal amounts of radioactivity were placed into the immune reaction I, without; 2, with 25 ug of purified SVS IV. Non-specific bands in the immunoprecipitates at 67, 43 and 35 kD are seen in both lane 1 and lane 2 and in the immunoprecipitates in fig. 8. Lane 3 contains 10 ug of purltied SVS IV and protein standards with their MWs indicated in kD. 1,2, Aurotadiographs; 3, Coomassie blue-stained.
DISCUSSION A method for establishing primary cultures of rat seminal vesicle epithelial cells has been reported, as well as the morphological and immunocytochemical characterization, of these cells in culture. During the course of this characterization, the contribution of androgen-priming to the synthesis and accumulation of one of the major proteins of the rat seminal vesicle has been evaluated. Results of this study show that epithelial cells from rat seminal vesicles can be isolated after
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dissociation with trypsin and collagenase and cultured under conditions in which structural and functional properties observed in the intact tissue are retained in culture. Although the structure and function of rat seminal vesicle epithelial cells have been evaluated in this report for a relatively short period of time (up to 4 days after plating), immunocytochemical and electrophoretic results support the assumption that this culture system will be useful for the study of androgendependent responses, including the synthesis and secretion of SVS IV. Previous studies have emphasized the use of primary cell cultures [23] or cell isolation procedures [15] to study androgen action on seminal vesicle cells. Lieber et al. [23] have established primary monolayer cultures of seminal vesicle cells from normal and castrated guinea pigs after collagenase dispersion of epithelium mechanically-stripped from the smooth muscle cell substrate. However, the immunocytochemical search of specific protein markers of guinea pig seminal vesicle epithelium [24] in these cultured seminal vesicle epithelial cells has yielded negative results. Epithelial and non-epithelial seminal vesicle cells from normal and castrated adult rats isolated after collagenase-pronase dissociation [15] were used to determine the synthesis and secretion of tissue-specific proteins in short-term (1 h) labeling experiments. Higgins et al. [ 151 have reported that isolated seminal vesicle cells synthesize and secrete several immunoprecipitable specific proteins similar to those of seminal vesicle tissue fragments incubated in vitro. Our in vitro approach is different from that of Higgins et al. [15] in that a relatively homogeneous population of seminal vesicle epithelial cells devoid of non-epithelial cell contaminants was allowed to attach to the culture substrate and further establish an epithelial-like growth pattern. Both cell attachment and growth, together with morphological preservation at the electron microscopic level, were taken as indications of cell viability. Similar criteria have been previously found to be reliable for establishing primary cultures of rat epididymal epithelial cells [25]. We were interested in evaluating the conditions required for the maintenance of differentiated cell function after attachment and growth of rat seminal vesicle epithelial cells. Based on the results of Ostrowski et al. [7], we induced with testosterone the synthesis and secretion of seminal vesicle proteins in sexuallyimmature, uncastrated rats and compared the results with non-induced, control animals. Both immunocytochemical and electrophoretic results show that androgen-priming of donor rats exerts a noticeable effect on the synthesis and accumulation of SVS IV in cultured seminal vesicle epithelial cells 3-4 days after plating. Under the present conditions, the only treatment that induced expression of SVS IV in cultures of premature seminal vesicle was in vivo priming of rats with androgen. The role of androgen in the maintenance of SVS IV production in these cultures remains to be determined. It is known, however, that the inclusion of androgen in the medium of cultures derived from premature, unprimed tissue does not induce expression of SVS IV during the 24 h labeling with [35S]methionine (data not shown). The immunocytochemical localization of SVS IV in epithelial cells cultured from rat seminal vesicles identifies them as in vitro counterparts of in vivo
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vesicle cell cultures
functioning cells. It can be argued, however, that SVS IV immunocytochemistry identifies a product of in vivo synthesis and subsequent storage. The immunoprecipitation of [35S]methionine-labeled product with specific antibody is evidence that seminal vesicle epithelial cells in vitro are capable of de novo synthesis of SVS IV. As mentioned previously, seminal vesicle cells retaining this expression of differentiated function in vitro were prepared from sexually-immature rats primed with androgen in vivo and maintained in the presence of a serum-free, androgen-supplemented medium during the 24-h [35S]methionine-labeling period. An intriguing aspect of this study is the finding that cultured seminal vesicle epithelial cells established from uncastrated, control rats secrete a protein precursor of slightly higher MW than purified SVS IV that is antigenically related to SVS IV. These two features as well as the hormonal status of the animal from which the cultures were obtained suggest that this protein may be a secreted form of SVS IV that has not undergone final processing. In view of the immunocytochemical and electrophoretic results reported in this paper, it is possible that the seminal vesicle culture system can be used to answer specific questions regarding the mechanism of induction, continued expression and perhaps processing of SVS IV, a marker of androgen-dependent differentiated cell function. We gratefully acknowledge the help of Dr Laura L. Tres in the isolation, culture and electron microscopic study of cultured seminal vesicle epithelial cells. We also thank Dr W. Stephen Kistler for making available to us purified SVS IV and rabbit anti-SVS IV serum as well as for stimulating discussions and critical review of the manuscript. This work was supported in part by grants HDl1884 (A.L.K.) and HD13472 (W. S. Kistler) from USPHS.
REFERENCES I. Cavazos, L F, Handbook of physiology, endocrinology (ed R 0 Creep & E B Astwood) vol. 5, p. 353. American Physiological Society, Washington, DC (1975). 2. Higgins, S J, Burchell, J M & Mainwaring, W I P, Biochem j 158 (1976) 271. 3. Barham, S S, Lieber, M M & Veneziale, C M, Invest urol 18 (1980) 13. 4. Ostrowski, M C, Kistler, M K & Kistler, W S, Biochemistry 21 (1982) 3525. 5. Harding, B W, Samuels, L T & Mann, T, Nat1 cancer inst monograph 12 (1963) 253. 6. Mansson, P-E, Carter, D B, Silverberg, A B, Tully, D B & Harris, S E, Nucleic acids res 6 (1979) 1553. 7. Ostrowski, M C, Kistler, M K & Kistler, W S, J biol them 254 (1979) 383. 8. Higgins, S J & Fuller, F M, Mol cell endocrinol 24 (1981) 85. 9. Gotterer, G, Ginsberg, D, Schulman, T & William-Ashman, H G, Nature 176 (1955) 1209. 10. Dravland, E & Joshi, M S, Biol reprod 25 (1981) 649. 11. Higgins, S J, Burchell, J M, Parker, M G & Herries, D G, Eur j biochem 91 (1978) 327. 12. Higgins, S J & Burchell, J M, Biochem j 174 (1978) 543. 13. Kistler, M W, Ostrowski, M C & Kistler, W S, Proc natl acad sci US 78 (1981) 737. 14. Bums, J M, Weinberger, M J & Veneziale, C M, J biol them 254 (1979) 2258. 15. Higgins, S J, Brooks, D E & Fuller, F M, Mol cell endocrinol 23 (1981) 207. 16. Rodriguez, J & Deinhardt, F, Virology 12 (1960) 316. 17. Kierszenbaum, A L, Feldman, M, Lea, 0, Spruill, W A, Tres, L L, Petrusz, P & French, F S, Proc natl acad sci US 77 (1980) 5322. 18. Spruill, WA, White, M G, Steiner, A L, Tres, L L & Kierszenbaum, AL, Exp cell res 131 (1981) 131. 19. Laemmli, U K, Nature 227 (1970) 680. 20. Cavazos, L F & Melampy, R M, Endocrinology 54 (1954) 640. 21. Tuohimaa, P & Miemi, M, Male accessory sex organs: structure and function in mammals (ed D Brandes) p. 329. Academic Press, New York (1974).
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Farquhar, M G & Palade, G, J cell biol 17 (1963) 375. Lieber, M M, Barham, S S & Veneziale, C M, Invest urol 17 (1980) 348. Veneziale, C M, Biochem j 166 (1977) 155. White, M G, Huang, Y-S, Tres, L L & Kierszenbaum, A L, J reprod ferti166 (1982) 475.
Received July 27, 1982 Revised version received December 29, 1982
Printed
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Cell
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145 (1983)
Isolation
Copyright @ 1983 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827183/060293-12$02.00/0
Cell Research 145 (1983) 293-304
and Culture
The Use of the Secretory
of Rat Seminal Protein
ABRAHAM L. KIERSZENBAUM, and IKUMASA TAKENAKA
Vesicle
Epithelial
SVS IV as a Functional
ROBERT
M. DePHILIP,
Cells
Probe
W. AUSTIN
SPRUILL
Department of Anatomy and The Laboratories for Reproductive Biology, School of Medicine, University of North Carolina, Chapel Hill, NC 27514, USA
SUMMARY A method for the isolation and culture of seminal vesicle epithelial cells obtained from control and androgen-primed sexually-immature, uncastrated rats is described. This method allows the establishment of monolayer cultures from aggregates of seminal vesicle epithelial cells isolated after trypsin and collagenase digestion. Phase contrast and transmission electron microscopic methods demonstrate that cell aggregates, after attaching to the substrate, establish within 48 h a colony-like, epithelial-like growth pattern. Immunofluorescent localization studies of SVS IV, an androgen-dependent secretory protein purified from rat seminal vesicle secretion, show that cultured seminal vesicle epithelial cells are immunoreactive. An electrophoretic analysis of [35S]methionine-labeled secretory proteins immunoprecipitated with rabbit anti-SVS IV serum demonstrate that, whereas SVS IV is newly-synthesized and accumulated in the medium of cultured seminal vesicle cells established from androgen primed rats, cultured cells from control rats appear to synthesize and accumulate SVS IV in a precursor form. Results of this work show that seminal vesicle epithelial cells in culture not only retain several structural features representative of the tissue but also serve as a potential system for the study of androgen action.
The seminal vesicle is an androgen-dependent accessory gland of the male reproductive tract. Under the influence of androgens, the seminal vesicle develops and maintains a secretory function. After castration, the organ undergoes involution (for a review see [l]) accompanied by a decline in its remarkable protein and secretory activity [2-4]. Secretory products of the rat seminal vesicle [5], including several proteins [2, 6-81, accumulate in the glandular lumen and are discharged into the urethra at the time of ejaculation. In rodents, one of the functional properties of seminal vesicle secretion is to contribute to clot formation of the ejaculated semen [9]. Although the direct functional role of seminal vesicle secretion on sperm fertility has not been established, it has been recently reported that a protein secreted by rat seminal vesicles binds to ejacuiated sperm
UOI. Major effort has been devoted to the characterization of rat seminal vesicle secretory proteins and their differential regulation by androgens [7, 8, 11, 121. It has been proposed that androgens control the synthesis and secretion of rat seminal vesicle proteins by regulating changes in the steady-state pool size for specific mRNA levels [4, 121. Ostrowski et al. [71 have reported five major rat seminal vesicle secretory proteins designated SVS I to V according to their increasing electrophoretic mobility. One of these proteins, SVS IV (corresponding to protein S of Higgins et al. [2]), has been regarded as a potential marker for androgen action [4, 71 and for assessment of the functional development of rat seminal vesicle [ 131. 20-838331
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The value of isolated epithelial fragments of seminal vesicles [14] or isolated epithelial cells of seminal vesicles maintained for short-term incubation experiments [15] has been reported. An alternative approach for long-term incubation experiments is the use of primary cell cultures of seminal vesicle epithelial cells. Primary cell cultures yield homogeneous populations of cells able to maintain, for a period of time, a degree of functional differentiation in a well-controlled environment. In this paper, we report a simple method for the isolation and culture of seminal vesicle epithelial cells from sexually-immature rats. The functional status of cultured seminal vesicle epithelial cells was evaluated by both immunocytochemistry and immunoprecipitation of [35S]methionine-labeled secretory proteins using a specific rabbit serum generated against SW IV. The combined use of these immunological approaches suggests a differential effect on SVS IV gene expression in cultured epithelial cells established from seminal vesicles of control and androgen-primed rats.
MATERIALS Isolation
AND
and culture
METHODS of rat seminal
vesicle epithelial
cells
Primary cultures of rat seminal vesicle epithelial cells were established from sexually-immature rats (Charles River CD) according to the procedure outlined below. The rats from which cultures were prepared varied from 28 to 35 days, the exact ages being included in the description of results. Seminal vesicles were removed under sterile conditions and coagulating glands were carefully removed under a dissecting microscope. The glands were minced with scissors into the smallest pieces possible and the luminal secretion washed out in Hanks Balanced Salt Solution (HBSS), pH 7.6. Tissue minces were dissociated with 0.25 % trypsin (Sigma T-8128) in HBSS (5 mg of tissue per ml solution) at 32°C for 30-40 min with continuous agitation in a thermo-bath shaker (60 cycles/min) in an Brlenmeyer flask. The sample was then allowed to settle for 5 min. The supernatant was discarded and the sediment suspended in a solution of collagenase (Sigma C-0130) 1 mg/ml in HBSS and incubated at 32°C for 90 min in a shaker as above. The sample was allowed to stand at room temperature for 5 min, the supematant discarded and the resulting sediment was resuspended in Eagle’s Minimum Essential Medium supplemented with 10% fetal bovine serum (Sterile Systems, Inc.), 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 4 mM glutamine, 100 U/ml penicillin and 100 ug/ml streptomycin. The sample, consisting of clusters of epithelial cells and single cells, was plated in tissue culture flasks, dishes or on glass and plastic coverslips (approximate cell density: 1 x lO?ml) and incubated in a humidified COz-air incubator at 32°C. Media were changed every 24 h. To minimize the possible contamination with non-epithelial cells (mainly smooth muscle cells and tibroblasts), the plated sample was allowed to settle in a tissue culture flask for about l-4 h. Under these conditions, non-epithelial cells attach very rapidly to the substrate while aggregates of seminal vesicle epithelial cells remain in suspension. The suspension of cell aggregates is then transferred to a new culture flask or dish where most of them attach within 24 h and establish an epithelial-like, colony-like growth pattern. Contamination with non-epithelial cells determined by phase contrast microscopy and transmission electron microscopy was less than 5 %. TO study the induction of SVS IV in vivo, sexually-immature male rats (21 or 28 days old) were given daily subcutaneous injections of 0.1 mg testosterone propionate in 0.1 ml sesame oil. Control rats were injected with sesame oil alone. After one week of treatment, where rats were either 28 or 35 days old, seminal vesicles were isolated for epithelial cell culture. Post-trypsin and post-collagenase samples were processed for transmission electron microscopy to monitor the enzymatic digestion of tissue (see below).
Transmission electron microscopy (TEM) Seminal vesicle epithelial cells cultured on plastic coverslips (Thermanox) were rinsed in phosphatebuffered saline (PBS) and fixed in 2.5 % glutaraldehyde in 0.1 M phosphate buffer (pH 6.9) for 1 h at room temperature and post-fixed in 1% osmium tetroxide in the same buffer. Specimens were embedded in Maraglas (Polysciences) according to standard procedure. Blocks were sectioned with
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orientations either parallel or perpendicular to the substrate. Post-trypsin and post-collagenase samples were fixed and embedded as described above. One urn thick sections were stained with toluidine blue. Thin sections (gray-silver transmitted interference color) were collected on Formvarcoated copper electron microscope grids and stained first with many1 acetate and then with lead citrate. Thin sections were examined with a JEM 1OOB transmission electron microscope.
Indirect
immunoj7uorescence
Seminal vesicle epithelial cells grown on glass coverslips and maintained in serum-supplemented Eagle’s Minimum Essential Medium were rinsed in PBS, fixed with 3.7 % formaldehyde for 15 min and permeabilized with 0.2% Triton X-100 in PBS for 2 min. Cells were incubated in 1.5% normal goat serum before and after incubation with rabbit anti-SVS IV serum for 1 h in a moist chamber at room temperature (working dilutions: 1: 25, 1 : 50 and 1 : 75). To localize bound SVS IV antibody, fluorescein isothiocyanate-conjugated goat anti-rabbit IgG was applied to coverslips for 30 min. Coverslips were then rinsed in PBS and mounted with Elvanol [16]. Immunocytochemical controls included: (1) omission of the primary antiserum; (ii) absorption of anti-SVS IV with immunoafftnity purified SVS IV [7] carried out as described [17, 181; (iii) immunoprecipitation of [35S]methioninelabeled seminal vesicle secretory proteins (see below); and (iu) immunohistochemical localization of SVS IV in the epithelium of intact seminal vesicles (not shown).
[“Slmethionine-labeling of rat seminal vesicle
of cultured epithelial
cells
Primary cell cultures plated in 25 cm* tissue culture flasks were incubated with [“Slmethionine (sp. act. 1 100 Cilmmol, New England Nuclear) for 24 h between the 2nd and 3rd day after plating. Just before labeling, cultures were rinsed two times with serum-free, hormone-supplemented medium. This medium consisted of Eagle’s Minimum Essential Medium containing l/10 the usual concentration of methionine and supplemented with the following to the final concentration indicated: nonessential amino acids, 0.1 mM; glutamine, 4 mM; sodium pyruvate, 1 mM; penicillin, 100 U/ml; streptomycin, 100 @ml; insulin, 5 &ml; transferrin, 5 &ml; epidermal growth factor, 3 &ml; human growth hormone, 6.5 @J/ml and retinol, 10 PM. Dihydrotestosterone (0.1 uM) was included in the medium of cells cultured from androgen-primed seminal vesicles. Each group of cells (control and medium containing 100 androgen-primed) was incubated in serum-free, hormone-supplemented t&i/ml [35S]methionine and, at the end of the labeling period, the media were collected and phenylmethylsulfonyl fluoride added to a final concentration of 10 mM. The media were centrifuged (13000 g) 15 min, 4°C and the supernatant frozen (-20°C). Incubation was carried out at 32°C in a humidified atmosphere containing 5 % CO*.
Immunoprecipitation
of [35S]methionine-labeled
SVS IV
A volume of medium from cell cultures containing 500000 trichloroacetic acid (TCA)-precipitable cpm was taken for specific immunoprecipitation. One-fifth this volume of 5x immune buffer (5x; 250 mM NaCl, 2.5 % sodium deoxycolate, 2.5 % Nonidet P-40, 2.5 % methionine and 100 mM Tris-Cl, pH 7.5) was added and the volume brought to 600 pl with lx immune buffer. Samples were preabsorbed with 100 ul of a 10% w/v suspension of protein A bearing Sruph. aureus cells (Pansobin, Calbiochem) for 30 min at 23°C. The Pansorbin was removed by centrifugation (5 000 g, 10 min, 4°C) and 30 ul of a 1 : 50 dilution of SW IV specific rabbit antiserum was added. Purified SVS IV (25 ug) was added to indicated samples to test the specificity of immunoprecipitation. Samples were incubated for 1 h at 37°C and then for 2 days at 4°C. Samples were brought to room temperature, 100 pl of Pansorbin added and incubation continued for 30 min at 23°C. Finally, the mixture was layered over a 1 ml cushion of immune buffer containing 1 M sucrose and the immunoprecipitate collected by centrifugation (5 000 g, 20 min, 4°C). The pellet was resuspended and washed two times with lx immune buffer. The final pellet was resuspended in 100 pl of electrophoresis sample buffer (10% w/v glycerol, 5 % v/v 2-mercaptoethanol, 2.3 % w/v sodium dodecyl sulfate (SDS) and 0.0625 M Tris-Cl, pH 6.8) [19] and heated at 100°C for 10 min. After cooling to room temperature, samples were clarified (13 000 g, 10 min, 23°C) and the supematant fractionated by sodium dodecyl sulfate-poiyacrylamide gel electrophoresis (SDS-PAGE) [19] on a 14x 14 cmx 1.5 mm slab gel of 15 % acrylamide overlaid with a 4 cm stacking gel of 4.75% acrylamide. After electrophoresis for 14 h at 100 V, gels were stained with Coomassie blue R and processed for fluorography using EN3HANCE (New England Nuclear) according to the procedure of the manufacturer.
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Fig. 1. Trypsin-collagenase dissociated seminal vesicle fragments of a 30-day old rat. The epithelium consists of simple columnar cells. The large cytoplasmic vacuoles are probably a consequence of tissue manipulation. Remnant of seminal vesicle secretion (SW) is present at the luminal epithelial side of the cell aggregate. Arrow indicates a contaminating non-epithelial cell. Inset. Enzymaticallydissociated seminal vesicle epithelium (as above) showing a mitotic cell (metaphase) with vesicles containing secretory granules (arrowheads). Arrow points to microvilli. CC, Golgi complex region. x 1920; (inset) x2 230.
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RESULTS Microscopic evaluation of enzymatically-dissociated vesicle epithelium
rat seminal
TEM studies of rat seminal vesicle tissue minces following trypsin and collagenase digestion demonstrate that most of the non-epithelial cell components of the stroma (mainly smooth muscle cells and tibroblasts) are removed (figs 1, 2) and that the samples, consisting of a predominant population of epithelial cells, retain the characteristic structural features reported in intact seminal vesicles [l]. Mitotic cells are occasionally observed (fig. 1). Seminal vesicle epithelial cells display a smooth basal surface which contrasts with the free apical region of the cell showing abundant microvillar extensions (fig. 2). Remnants of seminal vesicle secretion can be noted adjacent to the free apical cell region (fig. 1); Junctional complexes located at the apical, juxtaluminal cell region maintain the stability of epithelial cell clusters (fig. 2), a feature which fosters the establishment of a colony-like growth pattern after attachment of cell aggregates to the culture substrate (figs 3, 4). The characteristic topographic location of rough endoplasmic reticulum cisternae, Golgi complex regions and secretory vesicles, some of them displaying an eccentric electron-dense granule, are some of the most relevant cytoplasmic features observed in the seminal vesicle epithelium of control rats following enzymatic treatment (fig. 2). Similar features were observed in specimens from androgen-primed rats with the exception that the number of secretory vesicles was more abundant and the columnar epithelial cells taller (not shown). The cytological effects of testosterone propionate on the epithelium of rat seminal vesicles have been reported [20]. Morphological and immunocytochemical vesicle epithelial cells
evaluation of cultured rat seminal
After selective cell attachment of non-epithelial cells and re-plating of epithelial cell aggregates, as described in Materials and Methods, seminal vesicle epithelial cell aggregates from control and androgen-primed rats attach to the substrate within 24 h and establish an epithelial-like, colony-like growth pattern that is clearly apparent 48 h after plating (figs 3, 4). Primary cultures of seminal vesicle epithelial cells attain a confluent monolayer 5-7 days after plating. The size of the colony-like formations originating from cell aggregates of androgen-primed rats appear larger than those established from control rats (not shown). Because testosterone is known to induce in seminal vesicles of non-castrated rodents increased DNA synthesis accompanied by enhanced organ weight and DNA content in vivo [21], additional quantitative studies are required to determine in these cultures precise differences in cell number and volume, DNA synthetic activity and mitotic index between control and androgen-primed groups. Fig. 2. Detail of fig. 1 illustrating concentric arrays of rough endoplasmic reticulum cistemae (RER) at the basal cell region and the supranuclear location of Golgi complex saccules (CC) and secretory granules (arrozuheads). Arrows indicate linear densities corresponding to tight junctions between adjacent cells. MV, microvilli. x6 720.
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3. Phase contrast micrograph of seminal vesicle epithelial cells in a colony-like arrangement. indicates the central location of the originally-plated cell aggregate. 48 h after plating; 33-day old rat. x 330. Fig. 4. High magnification view of a colony-like formation of seminal vesicle epithelial cells displaying a characteristic epithelial-like organization. Phase contrast microscopy. 48 h after plating; 33-day old rat. X620. Fig. Arrow
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vesicle cell cultures
We wanted to determine general morphological features of cultured seminal vesicle epithelial cells after their adaptation to culture conditions. Primary cultures of control rats were studied by transmission electron microscopy 4 days after plating. Thin sections slightly oblique to the culture substrate (fig. 5) demonstrate that, although cultured seminal vesicle epithelial cells still retain numerous microvilli on the free apical cell surface (compare with figs 1 (inset), and 2), there is a considerable reduction in the size of secretory vesicles. Secretory vesicles located close to the microvillar cell region display an electrondense content (fig. 5). In general, the appearance, number and size of secretory vesicles in cultured cells differ from features observed in trypsin-collagenase tissue (fig. 2). An additional relevant feature observed in cultured seminal vesicle epithelial cells is the presence of tight junctions and desmosomes between adjacent cells (fig. 5). These junctional structures are regarded as indicative of epithelial cell nature [22]. As a result from the morphological analysis, it was assumed that, whereas seminal vesicle epithelial cells in culture lose some of the structural correlates of the intact seminal vesicle epithelium, they still maintained a certain degree of morphological differentiation. The next step in our study was the analysis of SVS IV immunoreactivity in cultured seminal vesicle epithelial cells established from control and androgenprimed rats using a specific rabbit anti-SVS IV serum. This study was carried out to further validate the epithelial nature of seminal vesicle cells in culture using a specific protein marker. A variable proportion of cultured seminal vesicle cells from 35day-old rats exhibited SVS IV immunoreactivity at the perinuclear region and scattered throughout the cytoplasm in the form of fluorescent spots (fig. 6A). This result is consistent with the fact that SVS IV is known to be detectable in sexually-immature animals of this age [13]. Under the same plating and culture conditions, almost all seminal vesicle cells cultured from androgenprimed rats displayed SVS IV immunoreactivity (fig. 6B-D). The specificity of SVS IV immunofluorescence was described in Materials and Methods. Synthesis of SVS IV in vitro We utilized SDS-PAGE to provide direct evidence that SVS IV is newly-synthesized by cultured seminal vesicle epithelial cells. Seminal vesicle secretory proteins from 28-day old rats that had received daily injections of testosterone propionate for 1 week were collected and fractionated by SDS-PAGE. Fig. 7 (lane I, Coomassie blue staining) shows that all the five major seminal vesicle secretory proteins [7] can be resolved. SVS IV is identified by its co-migration with purified SVS IV (fig. 7, lane 2, Coomassie blue staining). The seminal vesicles of control 28-day old rats, known to just begin to accumulate their secretory proteins [ 131, did not yield sufficient secretory product for electrophoretic analysis. Rabbit antibody specific for rat SVS IV was used to immunoprecipiFig. 5. TEM of cell components of a colony-like formation similar to fig. 4 (3 days after plating). A tight junction (arrou) and desmosomes (between crossed arrows) can be identified between adjoining cultured cells. The arrowheads indicate secretory vesicles near the microvillar (MV) cell region. CC, Golgi complex region; N, nucleus. x 14 400.
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6. Immunofluorescent localization of SVS IV in cultured rat seminal vesicle epithelial cells from control (35 days old); (B-D) androgen-primed (35 days old) rats. SVS IV immunoreactivity is more conspicuous in seminal vesicle cell cultures established from androgen primed rats. (D) Immunofluorescent granules which may correspond to the secretory granules depicted in TEMs (compare with fig. 5). The perinuclear immunofluorescence in the form of half-moon shaped patches (C) coexist with non-immunoreactive small vacoules. Dilution of anti-SVS IV serum: 1 : 75. (A, B) x370; (C) x375; (0) x 1280. Fig. (A)
tate [35S]methionine-labeled protein from the medium of epithelial cells derived from control and androgen-primed seminal vesicles (fig. 7, lanes 3 and 4, autoradiogram). We have found in medium from seminal vesicle epithelial cells cultured from control rats a very faint protein band co-migrating with SVS IV and a more intense protein band migrating more slowly than SVS IV (fig. 7, lane 3). Evidence that this higher MW band is antigenically related to SVS IV was provided by the ability of purified SVS IV to reduce the amount of radioactivity in this precipitated band (fig. 8, lane 2). A prominent [35S]methionine-labeled immunoprecipitated protein co-migrating with purified SVS IV was found in the medium of cultured seminal vesicle epithelial cells from androgen-primed rats (fig. 7, lane 4).
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67
Fig. 7. SDS-PAGE of seminal vesicle secretory proteins and SVS IV-specific immunoprecipitates from media of cells in culture. Secretory proteins in the seminal vesicle of a 28-day old, androgenprimed rat (lane I), purified SVS IV (lane 2) and [3’S]methionine-labeled, SVS IV-specific immunoprecipitates (lanes 3 and 4) were fractionated by SDS-PAGE on gels of 15 % a&amide following the procedure of Laemmli [ 191. The unlabeled secretory proteins (100 ug, lane 1) and purified SVS IV (10 pg. lane 2) were detected by Coomassie blue staining. [35S]Methionine incubation and immunoprecipitation were carried out as described in Materials and Methods. Equal amounts of radioactivity (5x 16 cpm) were added to the immunoprecipitation reaction. [35S]Methionine-containing proteins were detected by fluorography. 3, 4, Fluorographs of SVS IV-specific immunoprecipitates in the media of cells cultured from the tissue of 3, control; 4, androgen-primed rats. The five major seminal vesicle secretory proteins [7] are identified by Roman numerals and arrowheads. The positions of standard MW markers in kD are indicated on the right. Minor [35S]methionine-labeled protein bands in the immunoprecipitates presumably represent non-specific adsorption. Fig. 8. SVS IV-specific immunoprecipitation of [35S]methionine-labeled protein from the culture medium of cells from 28-day-old, untreated rats. Equal amounts of radioactivity were placed into the immune reaction I, without; 2, with 25 ug of purified SVS IV. Non-specific bands in the immunoprecipitates at 67, 43 and 35 kD are seen in both lane 1 and lane 2 and in the immunoprecipitates in fig. 8. Lane 3 contains 10 ug of purltied SVS IV and protein standards with their MWs indicated in kD. 1,2, Aurotadiographs; 3, Coomassie blue-stained.
DISCUSSION A method for establishing primary cultures of rat seminal vesicle epithelial cells has been reported, as well as the morphological and immunocytochemical characterization, of these cells in culture. During the course of this characterization, the contribution of androgen-priming to the synthesis and accumulation of one of the major proteins of the rat seminal vesicle has been evaluated. Results of this study show that epithelial cells from rat seminal vesicles can be isolated after
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dissociation with trypsin and collagenase and cultured under conditions in which structural and functional properties observed in the intact tissue are retained in culture. Although the structure and function of rat seminal vesicle epithelial cells have been evaluated in this report for a relatively short period of time (up to 4 days after plating), immunocytochemical and electrophoretic results support the assumption that this culture system will be useful for the study of androgendependent responses, including the synthesis and secretion of SVS IV. Previous studies have emphasized the use of primary cell cultures [23] or cell isolation procedures [15] to study androgen action on seminal vesicle cells. Lieber et al. [23] have established primary monolayer cultures of seminal vesicle cells from normal and castrated guinea pigs after collagenase dispersion of epithelium mechanically-stripped from the smooth muscle cell substrate. However, the immunocytochemical search of specific protein markers of guinea pig seminal vesicle epithelium [24] in these cultured seminal vesicle epithelial cells has yielded negative results. Epithelial and non-epithelial seminal vesicle cells from normal and castrated adult rats isolated after collagenase-pronase dissociation [15] were used to determine the synthesis and secretion of tissue-specific proteins in short-term (1 h) labeling experiments. Higgins et al. [ 151 have reported that isolated seminal vesicle cells synthesize and secrete several immunoprecipitable specific proteins similar to those of seminal vesicle tissue fragments incubated in vitro. Our in vitro approach is different from that of Higgins et al. [15] in that a relatively homogeneous population of seminal vesicle epithelial cells devoid of non-epithelial cell contaminants was allowed to attach to the culture substrate and further establish an epithelial-like growth pattern. Both cell attachment and growth, together with morphological preservation at the electron microscopic level, were taken as indications of cell viability. Similar criteria have been previously found to be reliable for establishing primary cultures of rat epididymal epithelial cells [25]. We were interested in evaluating the conditions required for the maintenance of differentiated cell function after attachment and growth of rat seminal vesicle epithelial cells. Based on the results of Ostrowski et al. [7], we induced with testosterone the synthesis and secretion of seminal vesicle proteins in sexuallyimmature, uncastrated rats and compared the results with non-induced, control animals. Both immunocytochemical and electrophoretic results show that androgen-priming of donor rats exerts a noticeable effect on the synthesis and accumulation of SVS IV in cultured seminal vesicle epithelial cells 3-4 days after plating. Under the present conditions, the only treatment that induced expression of SVS IV in cultures of premature seminal vesicle was in vivo priming of rats with androgen. The role of androgen in the maintenance of SVS IV production in these cultures remains to be determined. It is known, however, that the inclusion of androgen in the medium of cultures derived from premature, unprimed tissue does not induce expression of SVS IV during the 24 h labeling with [35S]methionine (data not shown). The immunocytochemical localization of SVS IV in epithelial cells cultured from rat seminal vesicles identifies them as in vitro counterparts of in vivo
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of SVS IV in seminal
vesicle cell cultures
functioning cells. It can be argued, however, that SVS IV immunocytochemistry identifies a product of in vivo synthesis and subsequent storage. The immunoprecipitation of [35S]methionine-labeled product with specific antibody is evidence that seminal vesicle epithelial cells in vitro are capable of de novo synthesis of SVS IV. As mentioned previously, seminal vesicle cells retaining this expression of differentiated function in vitro were prepared from sexually-immature rats primed with androgen in vivo and maintained in the presence of a serum-free, androgen-supplemented medium during the 24-h [35S]methionine-labeling period. An intriguing aspect of this study is the finding that cultured seminal vesicle epithelial cells established from uncastrated, control rats secrete a protein precursor of slightly higher MW than purified SVS IV that is antigenically related to SVS IV. These two features as well as the hormonal status of the animal from which the cultures were obtained suggest that this protein may be a secreted form of SVS IV that has not undergone final processing. In view of the immunocytochemical and electrophoretic results reported in this paper, it is possible that the seminal vesicle culture system can be used to answer specific questions regarding the mechanism of induction, continued expression and perhaps processing of SVS IV, a marker of androgen-dependent differentiated cell function. We gratefully acknowledge the help of Dr Laura L. Tres in the isolation, culture and electron microscopic study of cultured seminal vesicle epithelial cells. We also thank Dr W. Stephen Kistler for making available to us purified SVS IV and rabbit anti-SVS IV serum as well as for stimulating discussions and critical review of the manuscript. This work was supported in part by grants HDl1884 (A.L.K.) and HD13472 (W. S. Kistler) from USPHS.
REFERENCES I. Cavazos, L F, Handbook of physiology, endocrinology (ed R 0 Creep & E B Astwood) vol. 5, p. 353. American Physiological Society, Washington, DC (1975). 2. Higgins, S J, Burchell, J M & Mainwaring, W I P, Biochem j 158 (1976) 271. 3. Barham, S S, Lieber, M M & Veneziale, C M, Invest urol 18 (1980) 13. 4. Ostrowski, M C, Kistler, M K & Kistler, W S, Biochemistry 21 (1982) 3525. 5. Harding, B W, Samuels, L T & Mann, T, Nat1 cancer inst monograph 12 (1963) 253. 6. Mansson, P-E, Carter, D B, Silverberg, A B, Tully, D B & Harris, S E, Nucleic acids res 6 (1979) 1553. 7. Ostrowski, M C, Kistler, M K & Kistler, W S, J biol them 254 (1979) 383. 8. Higgins, S J & Fuller, F M, Mol cell endocrinol 24 (1981) 85. 9. Gotterer, G, Ginsberg, D, Schulman, T & William-Ashman, H G, Nature 176 (1955) 1209. 10. Dravland, E & Joshi, M S, Biol reprod 25 (1981) 649. 11. Higgins, S J, Burchell, J M, Parker, M G & Herries, D G, Eur j biochem 91 (1978) 327. 12. Higgins, S J & Burchell, J M, Biochem j 174 (1978) 543. 13. Kistler, M W, Ostrowski, M C & Kistler, W S, Proc natl acad sci US 78 (1981) 737. 14. Bums, J M, Weinberger, M J & Veneziale, C M, J biol them 254 (1979) 2258. 15. Higgins, S J, Brooks, D E & Fuller, F M, Mol cell endocrinol 23 (1981) 207. 16. Rodriguez, J & Deinhardt, F, Virology 12 (1960) 316. 17. Kierszenbaum, A L, Feldman, M, Lea, 0, Spruill, W A, Tres, L L, Petrusz, P & French, F S, Proc natl acad sci US 77 (1980) 5322. 18. Spruill, WA, White, M G, Steiner, A L, Tres, L L & Kierszenbaum, AL, Exp cell res 131 (1981) 131. 19. Laemmli, U K, Nature 227 (1970) 680. 20. Cavazos, L F & Melampy, R M, Endocrinology 54 (1954) 640. 21. Tuohimaa, P & Miemi, M, Male accessory sex organs: structure and function in mammals (ed D Brandes) p. 329. Academic Press, New York (1974).
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Received July 27, 1982 Revised version received December 29, 1982
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