Proteomic study of the establishment of boar epididymal cell cultures

Theriogenology 68 (2007) 76–86 www.theriojournal.com

Proteomic study of the establishment of boar epididymal cell cultures Judit Bassols a,*, Sergi Bonet a, Maya Belghazi b, Franc¸oise Dacheux c, Jean-Louis Dacheux c a

Biotechnology of Porcine Reproduction, Department of Biology, Faculty of Sciences, University of Girona, Campus de Montilivi s/n, 17071 Girona, Spain b Service de Spectrome´trie de Masse pour la Prote´omique, INRA, F-37380 Nouzilly, France c UMR 6175 INRA-CNRS, 37380 Nouzilly, France Received 19 January 2007; received in revised form 28 March 2007; accepted 1 April 2007

Abstract A proteomic approach was used in this study to follow the protein expression of epididymal cells during the different phases of a cell culture protocol which was able to obtain an epididymal cell monolayer. The secretory activity of intact proximal and middle caput epididymal fragments and caput, corpus and cauda epithelial cell monolayers was examined on different days of culture. Transcriptomic activity was also followed by RT-PCR for the mRNA of several previously identified major proteins. During the establishment of epididymal cell cultures, a progressive shift was found in the pattern of protein secretion. The normal epididymal protein profile, specific for each epididymal region, was progressively replaced by a less specific profile with the secretion of new proteins. A correlation between protein secretion and the presence of the mRNA of the marker proteins was observed only in the first phase of culture. Most of the new proteins which appeared were characteristic of the secretion of cell monolayers cultivated over several weeks. Despite the significant modifications of the epididymal cell secretome, the presence of new proteins secreted only by cell cultures originating from a specific epididymal region shows the presence of remaining endogenous differentiation. # 2007 Elsevier Inc. All rights reserved. Keywords: Boar; Epididymis; Cell culture; Proteins; Secretion

1. Introduction In mammals, the epididymis is the organ where spermatozoa become mature and are stored. The maturation stage of spermatozoa is the result of numerous interactions with external factors, mostly proteins, present in the lumen of the epididymal tubule. These proteins have several origins (testes, gametes and particularly the epididymis) where several hundred proteins are synthesized and sequentially secreted by

* Corresponding author. Tel.: +34 972418366; fax: +34 972418150. E-mail addresses: [email protected], [email protected] (J. Bassols). 0093-691X/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2007.04.009

the epididymal epithelium. The regional variations observed in the luminal proteins of several species are also the result of various modifications of these proteins throughout along the epididymis, such as reabsorption, sperm binding, proteolysis and post-transcriptional changes [1,2]. Most of these epididymal processes are dependent on androgens which are derived from the rete testis and blood circulation. The most active regulators responsible for maintaining epididymal structure and other epididymal functions are testosterone and dihydrotestosterone (DHT) [3]. Epididymal cultures allow direct evaluation of epididymal cell function, especially to elucidate the role and regulation of specific epididymal secreted proteins. Different in vitro approaches have been developed such as the initial

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studies using organ culture [4,5], individual epithelial cells [6], epithelial cell monolayers [7–13], in coculture with fibroblasts or spermatozoa [14–16], and immortalized epididymal epithelium [17–19]. In all these studies, the functions of cultured cells were evaluated by the presence of only certain specific epididymal mRNA or protein expression, and some studies using co-culture with spermatozoa have analyzed the effects of these proteins on sperm parameters such as motility, viability and sperm membrane composition. However, in all these epididymal cell culture systems, the comparison or the degrees of similarity with normal secretory protein activity of the epididymal epithelium have never been established. Prolonged culture of epithelial cells from different epididymal regions in the boar has recently been reported [20,21]. Furthermore, the secretory activity of the normal epididymal epithelium has previously been studied in this species, and the proteins secreted by the epididymal tubule have been characterized and most of them have been identified [22]. The aim of our study was to follow the transitory activity (or the evolution) of protein secretion between the epididymal cells in the epithelium and those in monolayers. We used a proteomic approach to monitor protein secretion from the proximal and middle caput epididymal fragments for 8 days, and then from the epididymal epithelial cell monolayers of the caput, corpus and cauda for 28 days. 2. Materials and methods

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(Rotiphorese Gel 30) from Carl Roth, GmbH I Co.; ampholytes pH 2–11 (Servalytes) from Serva (Heidelberg, Germany); and ampholytes pH 3–10 from GE Healthcare (Orsay, France). All other chemicals were of molecular biology grade and were purchased from Sigma. 2.2. Cell cultures Testes and epididymides were surgically obtained from three adult large white boars killed at a local slaughterhouse. The epididymides were removed from the testes and separated under sterile conditions into 10 regions, as previously described [23] (caput (Z0–Z4), corpus (Z5–Z7) and cauda (Z8–Z9)). For epididymal fragment cultures, epididymal tubules from the proximal (Z0) and middle caput (Z2) were dissected free of connective tissue and minced into small pieces (2– 5 mm) in RPMI-1640 medium containing 50 U ml1 of penicillin G and 50 mg ml1 streptomycin. After three washes with medium, about 10 fragments of each epididymal region were transferred to separate wells of a 48-well culture plate (Nunc, LabClinics, Barcelona, Spain) in 0.5 ml of epididymal culture medium (ECM) [21] and incubated at 32 or 37 8C in 5% CO2 in air and 100% humidity. However, in a preliminary experiment no difference was found when the tubules or the cells were incubated at 32 or 37 8C, so all the experiments were done at 37 8C. For epididymal epithelial cell cultures, tissue samples from the middle caput (Z2), middle corpus (Z6) and cauda (Z9) were collected, prepared in the presence of collagenase and cultured as previously described [20].

2.1. Chemicals RPMI-1640 medium, Dulbecco’s modified Eagle medium without methionine and cysteine (DMEM-), sodium pyruvate, N0 -2-hydroxyethylpiperazine-N0 ethanesulfonic acid (HEPES), penicillin–streptomycin, foetal bovine serum (FBS), trypsin–EDTA and Superscript II reverse transcriptase RNase H- were obtained from Invitrogen S.A. (France). Bovine insulin, hydrocortisone, testosterone, dihydrotestosterone, bovine apo-transferrin, retinol acetate, collagenase type VII, X-ray films (Kodak X-OMAT-XAR5; Eastman Kodak, Rochester, NY) and CHAPS (3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propanesulfonate) were purchased from Sigma Chemical Co. (St. Louis, MO); [35S] in vitro Cell Labeling Mix (Redivue PRO-MIX) from GE Healthcare (Orsay, France); synthetic oligo (dT) primer from Promega (France); DNA polymerase from Eurogentec (Seraing, Belgium); acrylamide

2.3. Secretion of [35S] methionine–cysteine-labeled proteins Intact tubule fragments cultured for 1, 2, 4, 6 and 8 days, and epididymal epithelial cell monolayers cultured for 7, 14, 21 and 28 days were washed three times in DMEM- and incubated for 4 h at 37 8C in DMEM- containing 100 mCi [35S] in vitro cell labeling mix. The incubating medium was then centrifuged (15,000  g for 5 min), and the supernatants were used or stored at 20 8C. 2.4. Gel electrophoresis The proteins present in the incubating medium of all cultures were separated by one- and two-dimensional electrophoresis gels, as previously described [22]. The gels were stained using the silver staining technique and

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Table 1 Oligonucleotide primers used in RT-PCR experiments Accession number

Protein name

Forward sequence

Reverse sequence

Q8SPJ0 O18994 Q29548 Q29549 O97763 P62936 Q9GKL7

Train A (RNase 10) GPX5 b-Hexosaminidase Clusterin NPC2 (HE1) Cyclophilin A Androgen receptor

50 -GAG-GAA-AGT-GAT-CAG-CTA-CTG-AGT-GAG-30 50 -GAC-GTG-ACA-GGC-ACC-ATC-TA-30 50 -TTC-CCT-GTG-ATC-CTT-TCT-GC-30 50 -CCA-GCC-CTT-CTT-CGA-CA-30 50 -GAG-AGG-AGG-GCA-TGA-GAA-TG-30 50 -TAA-CCC-CAC-CGT-CTT-CTT-30 50 -GCA-ACT-TCT-TCA-GCA-GCA-30

50 -GCT-CTG-AGC-ATC-TTG-TTT-CCT-CC-30 50 -GGG-AAA-GCC-CAA-CAC-AAC-TA-30 50 -TTG-CTC-GAG-GCC-ATA-GTC-TT-30 50 -CAG-AGT-GAT-GGG-GTA-GGA-30 50 -CCA-GCT-AGT-GGG-ATG-TGG-TT-30 50 -TGC-CAT-CCA-ACC-ACT-CAG-30 50 -GGG-TGC-TAC-ATC-GTC-CA-30

Train A = RNase 10, GPX = glutathione peroxidase, Hexo = b-hexosaminidase, HE1 = cholesterol transfer protein, Clus = clusterin, AR = androgen receptor, Cyclo = cyclophilin.

dried. Radioactive proteins were detected by autoradiography of pre-flashed X-ray film after several days’ exposure at 80 8C and by exposing the gels on a phosphor-imaging screen (GE Healthcare, Orsay, France). A representative protein pattern of each culture condition was obtained by comparing the autoradiograms of the three boars used for each experiment. Only spots and bands present in all the experiments were taken into account.

according to the supplier’s recommendations. Total RNA yields were estimated photometrically. Two micrograms of total RNA were reverse transcribed using Superscript II reverse transcriptase RNase H- and a synthetic oligo(dT) primer. The synthesized cDNA was amplified by DNA polymerase with 30 pmol of various pairs of primers detailed in Table 1. PCR was performed for 20–25 cycles (94 8C, 45 s; 55 8C, 45 s; 72 8C, 1 min) with a final elongation step at 72 8C for 5 min.

2.5. Protein identifications 3. Results Proteins were identified either by similarity between 2D electrophoresis separation and protein identified on an already published reference map of boar epididymal protein [22], or by mass spectrometry performed on 1D or 2D gel electrophoresis. Briefly, Coomassie bluestained bands or spots were excised from the gel and processed to obtain tryptic peptides according to a previously described method [24]. The tryptic fragments were sequenced by liquid chromatography combined with tandem Mass Spectrometry (nanoLCMS/MS) (Q-TOF-Global equipped with a nanoESI source; Waters Micromass) in automatic mode. The peptide masses and sequences obtained were either matched automatically to proteins in a nonredundant database (NCBI) using the Mascot program (http:// www.matrixscience.com) or de novo sequenced using the ProteinLynx Global Server program (Waters Micromass) and blasted manually against the current databases. 2.6. RNA extraction and RT-PCR assays Total RNA of intact epididymal fragments (cultured for 1, 2, 4, 6 and 8 days) and epididymal epithelial cells (cultured for 7, 14, 21 and 28 days) was extracted using an RNAble extraction kit (Eurobio, Les Ulis, France)

3.1. Morphological analysis of epididymal tubule cultures and establishment of cell cultures Intact epididymal tubule fragments were cultured for several days in ECM medium. Tubule appearance was maintained during the experiment. However, at day 8 of culture, no fragments or cells attached to the bottom of the culture well, in contrast to the epididymal fragments obtained after enzymatic digestion. In the latter preparation, epithelial cells migrated out of the tubule fragments and formed confluent monolayers in ECM after 12–14 days of culture. 3.2. Protein secretion 3.2.1. Protein secretion by epididymal tubule fragments The secretory activity of intact epididymal fragments of the proximal and middle caput were analyzed from day 0 to day 8 of culture to monitor the secretion of several major proteins characteristic of the epididymal regions and previously identified in the boar by Syntin et al. [22]. The synthesis and secretion of these proteins were analyzed by 1D (Figs. 1–3) and 2D (Fig. 2) electrophoresis gel separations.

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Fig. 1. SDS-PAGE and autoradiography of [35S] methionine-labeled proteins secreted in vitro by proximal and middle caput epididymal fragments after 0, 2, 4, 8 days of culture and by epididymal epithelial cell monolayers after 14 days of culture. A = Train A (RNase 10), G = glutathione peroxidase 5, H = b-hexosaminidase, C = clusterin. Numbered bands correspond to proteins identified by mass spectrometer (Table 2).

In the control samples incubated at day 0 (Fig. 1; Fig. 2A), the major secreted proteins (easily identified in the proximal caput and used as epididymal markers in our experiment) were glutathione peroxidase 5 (GPX5, 24 kDa), b-hexosaminidase (65 kDa) and Train A (RNase 10, 25–30 kDa). For the middle caput, clusterin (34–43 kDa), b-hexosaminidase (65 kDa) and HE1 (NCP2) (19–20 kDA) were chosen as markers (Fig. 2B). At day 2 of culture, these specific secretions were already reduced (Fig. 2C and D); at day 4 only traces were found (Fig. 2E and F), and at day 8 all the proteins characteristic of normal epididymal secretion had disappeared, except clusterin which was still traceable in middle caput culture (Fig. 2G and H). In contrast, some new proteins appeared during the culture in all the epididymal regions (Figs. 1 and 2). New serial spots of 43 and 90 kDa molecular mass appeared after 2 days of culture in the proximal caput cultures (Fig. 2C, arrows). These proteins became the major secretion at day 4 (Fig. 2E) and at day 8 they were reduced but still secreted. The particular positions of these proteins in 2D gel separation and the increase in molecular mass around 90 kDa in non-reducing conditions (Fig. 3) indicated that these proteins corresponded to certain isoforms of clusterin, previously identified by Syntin et al. [25]. However, the electrophoretic characteristics of these

new clusterin isoforms appeared different in their molecular mass shift according to the pI of those normally secreted by middle caput cells (Fig. 2B), suggesting that the glycosylation of these proteins could be different. 3.2.2. Protein secretion by epididymal epithelial cell monolayers The secretions of the cell monolayers obtained from caput, corpus and cauda epididymal fragments were analyzed by 1D and 2D gel electrophoresis after 7, 14, 21 and 28 days of culture. The protein secretion patterns obtained in 1D electrophoresis (Fig. 1) showed at least 20–25 proteins neosynthesized by the caput, corpus and cauda epithelial cell monolayers, but no differences were observed between regions or between different culture days (not shown). In 2D gel separation, several unidentified proteins secreted by epididymal epithelial cell monolayers coincided with the protein secreted by epididymal tubule fragments after 4–8 days of culture (Fig. 2, black cross). Among the proteins used as epididymal markers, only clusterin spots were detected in 14 days proximal caput epididymal epithelial cell cultures (Fig. 2I). No immunodetection with specific antisera against Train A, hexosaminidase, GPX5 or HE1 was obtained (data not shown). Using a more sensitive approach for protein detection by mass spectrometer, epididymal proteins

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Fig. 2. 2D SDS-PAGE and autoradiography of [35S] methionine-labeled proteins secreted in vitro by proximal caput (A, C, E, G) and middle caput (B, D, F, H) epididymal fragments after 0 (A, B), 2 (C, D), 4 (E, F) and 8 (G, H) days of culture and by epididymal epithelial cell monolayers originated from proximal caput (I) and middle caput (J) after 14 days of culture. GPX = glutathione peroxidase 5, Hexo = b-hexosaminidase, Train A = RNase 10, CLUS = clusterin, HE1 = NCP2 protein. Open circle corresponded to identified epididymal proteins and stars to proteins not secreted by epididymal cells according to Syntin et al. [22]. Black cross indicated common proteins between epididymal tubule fragments cultures and epididymal epithelial cell monolayers. The arrows indicated the proteins which appeared or disappeared.

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monitored by RT-PCR from day 0 to day 8 of culture (Fig. 4). The mRNA of Train A was present only in proximal caput cultures and the mRNA of GPX5, Hexo and HE1 was present in both proximal and middle caput cultures. All these mRNA could be detected at day 2 and then disappeared gradually. Clusterin mRNA was present in the middle caput during the whole culture period, and increased from day 2 in the proximal caput. Androgen receptor mRNA was present in both regions during the whole culture period. 3.3.2. Gene transcription in epididymal epithelial cell monolayers Expression of mRNA of clusterin, Hexo, HE1, Train A and GPX5 was analyzed by RT-PCR on epididymal cell monolayers from day 7 to day 21 of culture (Fig. 5). mRNA of clusterin, Hexo and HE1 was detected in caput, corpus and cauda epididymal epithelial cells. No differences were observed between epididymal regions or duration of culture. Train A mRNA was never detected in cell culture and GPX5 mRNA was found only in caput cultures at day 7. 4. Discussion

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Fig. 3. SDS-PAGE and autoradiography of [ S] methionine-labeled proteins in reducing (R) and non-reducing (NR) conditions, secreted in vitro by intact proximal (Z0) and middle (Z2) caput epididymal fragments after 8 days of culture. The arrows indicated the clusterin bands.

were sought in 1D gel bands cut from molecular mass in which epididymal proteins were likely to be located (Table 2). Several proteins were identified by MS/MS. Some of them, such as -N-acetylhexosaminidase, fructose–bisphosphate aldolase and calpain, were probably secreted but the expected epididymal proteins such as GPX5, HE1 were not identified by this technique. Most of the proteins found in the medium could be also related to the release of apoptotic epithelial cells. No proteins already known to be epididymal specific could be identified by this MS/MS approach. 3.3. Transcription of epididymal genes 3.3.1. Gene transcription in intact epididymal fragment cultures Expression of mRNA of Train A, GPX5, hexosaminidase, HE1, clusterin and androgen receptor from proximal and middle caput epididymal fragments was

Culture of the epididymal principal cells should provide a simplified system to study the regulation of cell activity and the interactions between spermatozoa and the epididymal epithelium. In vivo, study of the functions of epididymal epithelial cells is difficult because of numerous interactions such as peritubular cells, spermatozoa or substances present in the lumen of the tubule. Numerous methods have been proposed for cell culture of the epididymal epithelium and for all these methods, a cell monolayer was established from tubule fragments cultivated for several days, the time required to activate cell multiplication which is considerably reduced in the normal epididymal epithelium [26]. To evaluate the level of preservation of epididymal characteristics of these epididymal cultured cells, most studies have focused on the morphology of the cultivated cells, on the presence of epididymal messengers and specific proteins or sperm preservation of motility and fertility. None of these types epididymal culture has been evaluated by an approach which compares the overall protein secretion activity of these cells with those of the normal epididymal epithelium. An overall approach was used in this study to monitor the protein expression of epididymal cells during the different phases of a cell culture protocol which was able to provide an epididymal cell monolayer [20]. Protein

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Table 2 Protein detection by mass spectrometer of several bands present in 1D gels (Fig. 1) from epididymal epithelial cell monolayers after 14 days of culture Band number

Accession number

Proteins

% Sequence coverage

Mascot score

MM

Band 1 (41 kDa)

P60712 P08729 P05786 Q5BJY9 P48616 P11979 P08865

Actin, cytoplasmic 1 Cytokeratin-7 Cytokeratin-8 Cytokeratin-18 Vimentin Pyruvate kinase 34/67 kDa laminin receptor

63 13 26 13 8 5 11

726 308 282 171 86 68 53

41,710 51,241 42,369 47,601 53,484 57,878 32,702

Band 2 (39 kDa)

P08728 P60712 P04075 Q7SIB7

Cytokeratin-19 b-Actin Fructose-bisphosphate aldolase A Phosphoglycerate kinase 1

41 40 63 11

849 448 90 66

43,858 41,710 39,264 44,399

Band 3 (29 kDa)

P63103 Q6QRN9 P35232 Q58DT1 P18669 P62701 Q29548 P08166 P06813 P30041 Q5E947 P02067

Protein kinase C inhibitor protein 1 ADP/ATP translocase 3 Prohibitin 60S ribosomal protein L7 Phosphoglycerate mutase 1 40S ribosomal protein S4 b-N-acetylhexosaminidase Adenylate kinase isoenzyme 2 Calpain small subunit 1 1-Cys peroxiredoxin Peroxiredoxin 1 Hemoglobin b subunit

52 28 55 30 24 7 5 10 13 13 5 85

468 343 265 195 98 88 68 66 51 49 46 431

27,728 32,758 29,786 29,150 28,655 29,448 61,011 26,349 28,221 24,888 22,195 16,024

Band 4 (18 kDa)

P01965 P62808 P62802 P04908 Q71LE2

Hemoglobin a subunit Histone H2B Histone H4 Histone H2A type 6 Histone H3.3

78 52 47 35 8

374 222 194 165 49

15,030 13,767 11,229 14,033 15,187

secretions were thus followed from the epididymal cells still included in the epithelium to the cell monolayer formed by migrated cells out of the epithelium. Transcriptomic activity of these cells was monitored by RT-PCR of several mRNA of major proteins previously identified in the boar [27–29] such as messengers of hexosaminidase, glutathione peroxidase, HE1 and Train A. We found that the specific secretion of the epididymal tubule cultivated in vitro was partially preserved during the first two days of culture. A previous study showed that the supernatant of the cultured tubule was able to promote modifications in immature sperm [21]. The protein secretion of the tubule fragments changed after these first two days with progressive loss of epididymal specificity. After a week in culture, all the specific epididymal proteins were undetectable. However, the rapidity of the disappearance of protein secretion was protein dependent, RNase 10 and GPX5 being the most sensitive of the proteins identified.

This decrease in protein secretion was parallel to a similar decrease in the mRNA concentration of for example RNase 10 (Train A), GPX5, HE1 and hexosaminidase. However, this correlation was only observed when the epididymal cells were still in their own epithelium. When the epididymal cells formed monolayers, the correlation between mRNA and protein secretion was not observed for all the marker proteins, since mRNA for HE1 and hexosaminidase was again detected without evidence of the corresponding proteins. This discrepancy may be linked to the epididymal specificity of the proteins, RNase 10 and GPX5 being specific epididymal proteins [30,31] although HE1 and hexosaminidase are also described as lysosomal proteins [32]. As the principal epididymal cells are highly polarized, with an apical surface in contact with a specific luminal fluid, this rapid disappearance of the secreted proteins may be linked to the fact that a single medium surrounded both apical and basal parts of the tubule in our experiment. However, the same disappearance of secretory activity

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Fig. 4. RT-PCR analysis of various genes encoding epididymal proteins in intact proximal and middle caput epididymal fragments cultured for 0, 2, 4 and 8 days. Cyclophilin was used as control. Train A = RNase 10, GPX = glutathione peroxidase 5, Hexo = b-hexosaminidase, HE1 = NCP2 protein, Clus = clusterin, AR = androgen receptor, Cyclo = cyclophilin.

was found when culture was performed with closed epididymal fragments which preserved the original luminal content of the tubule (unpublished results). Histological examination of these tubules in culture showed that the integrity of the principal cells in the epididymal epithelium was progressively disrupted, in spite of good preservation of the peritubular cells which kept the structure of the tubule intact. Rapid decrease has been also described for an epididymal protein marker (CP 24) in the mouse, after 3–4 days in epididymal cell culture [15]. The presence of fibroblasts co-cultivated with isolated epididymal cells in this study, delayed the decrease but did not prevent the loss of secretion of this marker after 8 days, suggesting that mesenchyme-derived cells play a transitory role in the maintenance of epididymal cells [15]. The presence of peritubular cells in our tubule fragment cultures which might also have a potentially efficacious beneficial role, was not sufficient to maintain the normal phenotype of epididymal epithelial cells.

Although, specific epididymal proteins disappeared from the secretome in tubule fragment cultures, new proteins not normally secreted in vivo by the epididymal tubule appeared in the supernatant. Most of these new proteins were found to be still secreted by the cell monolayers. These new proteins were detected after 4 days of tubule culture whatever the epididymal origin of the tubule. The number of proteins and their secretion intensity increased during culture. Moreover, these proteins were secreted progressively whereas the typical epididymal proteins progressively disappeared. None of them have been yet identified, but their presence probably illustrates dedifferentiation steps of these epithelial epididymal cells. Among these new proteins, one appeared sooner than the others in the culture medium and was secreted specifically by the tubule fragments originating from the anterior caput of the epididymis. This protein, identified as the clusterin, was secreted in high quantities at day 4 of culture and was still present in

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Fig. 5. RT-PCR analysis of various epididymis-expressed genes in caput (Ct), corpus (Cp) and cauda (Cd) epididymal epithelial cell monolayers after 7, 14 and 21 days. RNA extracted from caput, corpus and cauda epididymides was used as in vivo control (day 0). Cyclophilin was used as control. Train A = RNase 10, GPX = glutathione peroxidase 5, Hexo = b-hexosaminidase, HE1 = NCP2 protein, Clus = clusterin, AR = androgen receptor, Cyclo = cyclophilin.

the monolayer cells after 14 days. None of the tubule fragments of the other epididymal regions showed this activation of clusterin secretion, even those which normally secreted it such as in the posterior caput. The anterior caput of the epididymis is a particular region with specific features, both in the nature of the proteins secreted such as RNase 10 and in its regulation factors, mostly originating from testis [22]. In vivo, clusterin secretion is activated in this region when an animal is castrated [25] because clusterin is one of the epididymal proteins whose transcription is suppressed by androgens [33]. However, in our experiment, the presence of androgen in the culture medium did not prevent clusterin secretion. Clusterin secretion has often been related to the presence of cell apoptosis [34] which was seen in our culture system. However, no increase in clusterin secretion was found in the cultivated fragments from other epididymal regions which presented similar signs of apoptosis. Thus, the proximal caput epididymis might specifically regulate the clusterin gene, probably in combination with unknown direct or indirect testicular factors.

The fact that clusterin was secreted only in the cell monolayer originating from the anterior caput region proved that these cells retain a regional epididymal characteristic although the normally secreted major epididymal proteins could not be detected by 2D gel electrophoresis. Many proteins were identified by mass spectrometry and the numerous cytokeratins confirmed that cell monolayers had an epithelial origin. None of the other proteins identified corresponded to the known major epididymal proteins secreted. In conclusion, there is a progressive shift in the pattern of protein secretion during the different phases of the establishment of epididymal cell cultures. The normal epididymal protein profile which is specific for each epididymal region, is progressively replaced by a less specific profile with new protein secretions. Most of these new proteins characterize the secretion of monolayer cells cultivated over several weeks. These important changes in the epididymal cells secretome and the presence of new proteins secreted by cell cultures originating from a specific epididymal region are evidence of endogenous dedifferentiation. The

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highly specific epididymal proteins (such as RNase 10 and GPX5 in our study) have never been reported to be secreted in epididymal cell culture, whatever the culture method used. A complete transcriptomic study similar to this proteomic approach should be very informative but the discrepancy between transcriptome and proteome strengthen that the proteomic approach is an indispensable step to characterize the functionality of the highly specialized and regulated epididymal epithelium, and provide a real possibility for epididymal physiology studies in vitro. Acknowledgements This study was made possible by a grant from ‘‘Estancias breves en el extranjero y en Espan˜a para becarios del Programa Nacional de Formacio´n de Profesorado Universitario’’ of the Ministerio de Educacio´n y Ciencia (MECD). We thank G. Tsikis for technical assistance, B. Delaleu for ultrathin sections and E. Venturi for providing the animals.

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