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Hormonal Regulation of Bovine Secretory Proteins Derived From Caput and Cauda Epididymal Epithelial Cell Cultures

ALAN K. GOFF

From the ^* Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium; and the CRRA, Faculty of Veterinary Medicine, University of Montréal, St-Hyacinthe, Canada.

Correspondence to: Dr Ann Van Soom, Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium (e-mail: ann.vansoom{at}rug.ac.be).

Received for publication September 10, 2002; accepted for publication January 10, 2003.

	Abstract

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The goal of this study was to investigate the effect of hormones (testosterone, dihydrotestosterone [DHT], and hydrocortisone) on the protein secretion of caput and cauda epididymal epithelial cells cultured in principal cell medium (PCM). A confluent monolayer of caput and cauda epididymal epithelial cells was obtained from serum-containing PCM in the presence or absence of hormones after 7 days of culture at 38.5°C (5% CO₂ in air). The protein secretion of epididymal epithelial monolayers incubated in serum-free PCM for 3 days was examined. The secreted proteins were separated by 2-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D SDS-PAGE). A comparison of the different protein patterns showed 61 spots, of which 11 were secreted only in the presence of hormones, 3 appeared to show hormone-related changes, and 25 were region-specific. Most of these secreted proteins were low-molecular-weight acidic proteins. To obtain evidence of the epididymal origin of the secreted proteins, proteins present in caput and cauda epididymal plasma were analyzed. In conclusion, our data indicate that hormones influence the synthesis of a number of caput and cauda epididymal proteins. Some of these proteins could be important for improving our understanding of spermatozoa maturation and storage and their acquisition of fertilizing ability.

     Key words: Epididymis, androgens

Recently, prolonged cultures of epididymal cells have also been established in cattle (Gagnon et al, 2000; Reyes-Moreno et al, 2000). Moreover, it has been shown that the motility of frozen-thawed spermatozoa was partially preserved for 48 hours after coculture with caput, corpus, or cauda epididymal cells, whereas conditioned medium (Gagnon et al, 2000) or bovine epididymal plasma (Reyes-Moreno et al, 2002) was effective in preserving sperm motility for only 6 hours. It was concluded that epididymal epithelial cells secrete one or more beneficial compounds, which prolong sperm viability. This compound must be a common factor present in epididymal plasma and secreted by epididymal epithelial cells cultured in vitro. A number of epididymal proteins may be involved in the protection of ejaculated sperm during in vitro storage (Reyes-Moreno et al, 2002). It was the purpose of our study to investigate which proteins

1. are influenced by the presence of testosterone, DHT, and hydrocortisone in the culture medium;
   
2. are common to conditioned media of caput and cauda epididymal epithelial cell cultures; and
   
3. are common to conditioned media of caput or cauda epididymal cell cultures and epididymal plasma.
   

	Materials and Methods

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Medium

Principal cell medium (PCM) consisted of RPMI-1640 medium (Life Technologies, Gibco BRL Products, Merelbeke, Belgium) supplemented with 10% fetal calf serum, 100 nM insulin, 1 mM sodium pyruvate, 5 µg/mL transferrin, 200 nM hydrocortisone, 200 nM testosterone, 1 µM DHT, 1 µg/mL retinol, and 50 µg/mL gentamicin. Testosterone and DHT were used because they control the synthesis and secretion of a number of specific proteins by the epididymis and maintain the epididymal structure and functions (Robaire and Viger, 1995). Hydrocortisone promotes cell attachment (Ballard and Tomkins, 1969) and cell proliferation (Guner et al, 1977). PCM without hydrocortisone, testosterone, and DHT was used as the control.

Collection of Caput and Cauda Epididymal Plasma

Epididymides of beef bulls aged 2–2.5 years were collected at a local slaughterhouse. After removing superficial blood and tissue fluid contamination, the pressure in the epididymal ducts was increased by clamping 2 pincers on the proximal and distal parts of the caput and cauda epididymidis. Incisions were made in the connective tissue, avoiding small blood vessels. The epididymal plasma oozed out and was aspirated into a fine pipette and transferred into a small tube. After 5 minutes of centrifugation at 3.214 x g, the epididymal plasma was removed, checked visually for the absence of spermatozoa, and frozen at -20°C.

Epididymal Epithelial Cell Culture

The epididymal epithelial cell culture was prepared by a modified protocol according to Moore et al (1986). The epididymal tissue was obtained from bulls slaughtered in a local slaughterhouse. On arrival in the laboratory, the caput and cauda epididymidis from 1 bull were dissected free of the testis, fat, and connective tissue and washed in RPMI-1640 medium to remove blood. Both regions were minced in small segments of 1–2 mm using scissors and placed in PCM (Moore et al, 1992). The spermatozoa within the tubule segments were teased out using forceps. The tubule segments were then incubated in PCM containing 1.5 mg/mL collagenase type II (Sigma Chemical Co, Bornem, Belgium) at 38.5°C in 5% CO₂ in air for 2 hours. After enzymatic digestion, tubule segments were dissected free of surrounding collagen with a needle, washed again in PCM to remove remaining spermatozoa, and transferred to fresh medium. They were slit open longitudinally and cut into small fragments. These fragments were prepared from both caput and cauda epididymidis and cultured separately in PCM supplemented with fibronectin (2 µL/mL) (Sigma) and in PCM without hormones and supplemented with fibronectin (2 µL/mL) (control) at 38.5°C and 5% CO₂ in air. The culture medium was changed every other day. The fragments formed irregular contiguous spheres of epithelial cells with the apical surface facing outward and remained free-floating in culture during the first days. These spheres became attached to the dish, and the epithelial cells were spread out (Moore et al, 1992). The attached explants were carefully removed with a 26-gauge needle connected to a 1-mL syringe. The epithelial cells continued to divide, forming a monolayer after 5–7 days of culture. The medium was then replaced by serum-free PCM with or without hormones. After 3 days of incubation, cultures were examined for epithelial cell detachment by means of inverted light microscopy. The epithelial and fibroblast cell concentrations of the monolayer were examined by immunohistochemistry. The medium was collected from the different groups and stored at -20°C until analysis by 2-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D SDS-PAGE).

Immunohistochemistry

Monolayers cultured on glass coverslips were rinsed with phosphate-buffered saline (PBS), fixed for 10 minutes in acetone, and air dried. After rinsing the slides 3 times with PBS, the cells were incubated for 2 hours with an anticytokeratin antibody (Keratin Pan Ab-1, NeoMarkers, Labvision, Fremont, Calif) diluted 1:500 in PBS at 37°C to establish the proportion of epithelial cells (Henriksen et al, 1990). The cells were then washed for 10 minutes in PBS and incubated with a biotin-conjugated goat anti-mouse immunoglobulin antibody for 30 minutes at room temperature. After washing the slides in PBS for 10 minutes, the cells were incubated with Strept-ABComplex/horseradish peroxidase for 30 minutes at room temperature. Horseradish peroxidase activity was visualized by incubating the slides with 3,3'-diaminobenzidine tetrahydrochloride (DAB tablets, Sigma) and 0.5 mg/mL Tris-HCl buffer, pH 7.6, containing 0.02% hydrogen peroxide for 10 seconds, which resulted in brown staining. After washing, cells were counterstained with Mayer Hemaluin solution (VWR International, Leuven, Belgium), washed, coverslipped, and viewed with light microscopy (Leica DMR, Van Hopplynus NV, Brussels, Belgium). Another replicate of epididymal cultures was probed with antibody against vimentin (vimentin Ab-2 [V9], NeoMarkers, Labvision) to establish the proportion of fibroblasts.

The epithelial and fibroblast cell concentration was measured with the Image Database Program of Leica.

2D Gel Electrophoresis

Before the separation and analysis of the proteins by means of 2D SDS-PAGE, the proteins of the conditioned media were concentrated using Ultrafree-15 concentrators (5000-MW cutoff; Millipore, Bedford, Mass). The protein concentration of the different samples was measured using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Richmond, Calif). Proteins were then added to an IPG buffer (8 M urea, 2% CHAPS, 0.5% IPG buffer [pH 3–10], bromophenol blue, and 65 mM dithiothreitol [DTT]) to give a final concentration of 2 µg/100 µL IPG buffer. The separation in the first dimension was carried out using Immobiline DryStrips (Amersham Pharmacia Biotech AB, Baie d'Urfé, Canada) that had been rehydrated in 250 µL of the sample/IPG buffer solution (5 µg total protein) for at least 10 hours in an Immobiline Drystrip Reswelling Tray (Amersham). The samples were then separated on a MultiPhore II flatbed system (Amersham) for 16 hours at 15°C. The voltage was 300 V for the first 3 hours, from 300 to 2000 V for the following 5 hours, and finally, 2000 V for 8 hours. Before the second dimension was performed, the dry strips were equilibrated for 10 minutes in Equilibration solution 1 (0.5 M Tris/HCl, pH 6.8, containing 0.36 g/mL urea, 10 mg/mL SDS, 2.5 mg/mL DTT, and 26% glycerol) and for another 10 minutes in Equilibration solution 2 (0.5 M Tris/HCl, pH 6.8, containing 0.36 g/mL urea, 10 mg/mL SDS, 45 mg/mL iodoacetamid, and 26% glycerol). The second dimension was performed after placing the strips on Pharmacia ExcelGel XL SDS 12–14 using the MultiPhore II flatbed system for 3–4 hours at 15°C. After the gels were run, they were immediately immersed in fixing solution (50% methanol and 10% acetic acid in water) and stained with silver nitrate (Silver Staining Kit, Amersham). The gels were compared, and the molecular weight (MW) and isoelectric point (pI) of the proteins were calculated using 2D SDS-PAGE analysis software (Phoretix, New-castle-upon-Tyne, England).

This experiment was repeated with 3 different caput and cauda epithelial cell cultures. A representative protein pattern of each culture condition was obtained by comparing scans of 3 gels of each culture, and only spots present on the 3 gels were taken into account. The concentration of each spot was expressed as its spot volume, which is the product of the area of the spot and its total optical density. Differences in spot density between cultures were determined by the Student's t test.

	Results

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Formation of Cell Cultures

A confluent monolayer of the caput and caudal part of the epididymis was successfully obtained after 5–7 days of culture in serum-containing PCM at 38.5°C in 5% CO₂ in air with or without the presence of hormones. From day 7 until day 10, the medium was replaced by serum-free PCM with or without hormones. Under all the cell culture conditions, there was no epithelial cell detachment for the first 10 days in culture. Primary cell cultures of caput and cauda epithelial cells can be maintained for up to 10 days without substantial overgrowth of fibroblasts, as determined with antibodies to vimentin. More than 90% of the cells in the monolayer were epithelial cells, as identified by the presence of cytokeratin (data not shown). No significant difference in the mean epithelial cell concentration of 3 replicates was observed between hormone-containing cultures (caput = 398 040 epithelial cells/1.77 cm²; cauda = 346 680 epithelial cells/1.77 cm²) and hormone-free cultures (caput = 369 792 epithelial cells/1.77 cm²; cauda = 333 840 epithelial cells/1.77 cm²) (ANOVA, SPSS 10.0 for Windows, SPSS Inc, Chicago, Ill).

Protein Analysis Using 2D SDS-PAGE

A comparison of the different protein patterns showed 61 proteins secreted by epididymal epithelial cell cultures. Some of these proteins were induced or down-regulated, or the concentration was increased or decreased in the presence of hormones (Table). Forty-seven of these proteins were also observed in caput or cauda epididymal plasma (Figure 1).

View larger version (98K): [in this window] [in a new window]   Figure 1. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of proteins of epididymal plasma. Phoretix 2D advanced software was used to detect and compare protein spots. The marked spots represent proteins that correspond to those found in the medium after culture of caput or cauda epithelial cells (see Figures 2 and 3). (A) Plasma from the caput region. (B) Plasma from the cauda region.

View larger version (60K): [in this window] [in a new window]   Figure 2. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of proteins secreted from caput epididymal cells. Phoretix 2D advanced software was used to detect, compare, and calculate the volume of protein spots. Only spots found on 3 different replicates were labeled, and all spots were numbered sequentially, starting with the highest molecular weight (MW). Gels are shown for epithelial cells cultured in principal culture medium (PCM) (caput) and epithelial cells cultured in PCM plus hormones (200 nM testosterone, 1 µM dihydrotestosterone, and 200 nM hydrocortisone) (caput H). Spots marked with a circle are common to both the control and hormone-treated cultures, and spots marked with a square are found only in the control or only in the hormone-treated cultures.

View larger version (63K): [in this window] [in a new window]   Figure 3. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of proteins secreted from cauda epididymal epithelial cells in culture. Phoretix 2D advanced software was used to detect, compare, and calculate the volume of protein spots. Only spots found on 3 different replicates were labeled, and all spots were numbered sequentially starting with the highest molecular weight (MW). Gels are shown for epithelial cells cultured in principal culture medium (PCM) (cauda) and epithelial cells cultured in PCM plus hormones (200 nM testosterone, 1 µM dihydrotestosterone, and 200 nM hydrocortisone) (cauda H). Spots marked with a circle are common to both the control and hormone-treated cultures, and spots marked with a square are found only in the control or only in hormone-treated cultures.

The effect of hormones on protein secretion from cauda epididymal epithelial cells is presented in Figure 3. At least 50 proteins were secreted into the conditioned media, of which 30 were also observed in cauda epididymal plasma. Of the 30 proteins found in both the control and hormone-treated samples, the intensity of 3 was significantly (P < .05) altered by hormones. The intensity of protein 4 was increased in the presence of hormones, whereas that of proteins 10 and 50 was decreased. The secretion of 20 proteins with a pI value in the range of 4–6.5 was regulated by hormone treatment: 4 proteins (spots 3, 23, 25, and 27) were induced by hormones, and 16 proteins (spots 5, 7, 9, 18, 24, 26, 32, 33, 34, 35, 47, 51, 52, 56, 59, and 61) were down-regulated.

Differences in protein composition secreted by epididymal epithelial principal cells from the caput vs cauda epididymidis were also observed (Figure 4). Of the 61 proteins that were consistently present, 11 (spots 15, 19, 28, 31, 36, 37, 45, 55, 62, 63, and 64) were secreted only by caput epithelial cells, while 14 (spots 3, 5, 6, 9, 21, 22, 24, 25, 26, 27, 29, 33, 35, and 54) were unique to cauda epithelial cell cultures. Thirty-six proteins were secreted by both caput and cauda epididymal epithelial cells.

View larger version (16K): [in this window] [in a new window]   Figure 4. General master pattern summarizing the total number of protein spots from media of control or hormone-treated caput and cauda epididymal epithelial cells. Spots marked with a triangle are secreted only in control or hormone-treated caput epididymal epithelial cell cultures; spots marked with a square are secreted only in control or hormonetreated cauda epididymal epithelial cell cultures; and spots marked with a circle are common to both caput and cauda cell cultures.

	Discussion

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In cattle, little is known about the processes through which the epididymal epithelium and its secretions maintain sperm viability in vitro. These processes appear to be mediated by proteins secreted by the epididymal mucosa under the influence of androgens. Although epididymal cell culture or even conditioned medium (both in the presence of androgens) can be used to improve bovine sperm survival and/or motility in vitro (Gagnon et al, 2000; Reyes-Moreno et al, 2000), this approach is not a practical alternative for the development of a diluter for prolonged storage of fresh bovine semen.

Evidence for the viability and normal differentiation of our caput and cauda epididymal epithelial cell cultures was given by the fact that both regions displayed the same capacity to form a confluent monolayer of epithelial cells in PCM and remained viable in vitro for at least 10 days. In contrast to the results of Gagnon et al (2000) and Moore et al (1992), no signs of cell degeneration, reduced growth, or contamination with fibroblasts were apparent in caput and cauda epididymal epithelial cell cultures in the absence of hormones. The cultured epithelial cells exhibited less than 10% contamination with other cell types, as determined by staining with cytokeratin. This percentage was similar to the observations made by Gagnon et al (2000).

To improve our insight into the molecular mechanism of sperm protection, we have focused our attention on proteins present in conditioned media from epididymal cultures of caput or cauda epithelial cells in the presence or absence of hormones. We have shown that 66% and 60% of the proteins present in caput or cauda conditioned media, respectively, correspond to proteins secreted in caput or cauda epididymal plasma, which confirms their epididymal origin. Proteins present in the conditioned media while absent in the epididymal plasma could correspond to proteins that are integrated into the sperm plasma membrane during epididymal transit or to those that are degraded or reabsorbed rapidly after secretion.

The majority of the secreted proteins had molecular masses between 20 and 80 kd and acidic pI values between 4 and 7. Besides the hormone dependency of protein synthesis by the epididymal cells, substantial differences in protein patterns from caput and cauda epididymal regions could be detected (Figure 4). In addition to proteins that were induced uniquely by hormones, the secretion of only a few proteins was either enhanced or reduced by these hormones.

The precise function of these secreted proteins in sperm maturation and storage processes remains to be established. Indirect evidence for a function can come from their relative abundance and from their site-specific expression along the epididymal duct. A further approach could be the identification of some major epididymal proteins by means of protein microsequencing, recombinant DNA techniques, or specific antisera (Syntin et al, 1996) or by investigating their sequence similarities to proteins or protein families of known functions and their immunolocalization on the sperm surface (Kirchhoff, 1998). The concentration of a protein present in epididymal plasma from different regions is not necessarily related to its level of secretion (Syntin et al, 1996). Proteins present at low concentrations in epididymal plasma may be those that are degraded, reabsorbed, or integrated into the sperm membrane (Syntin et al, 1996) and thus are possibly of greater interest than more abundant proteins. This is in contrast with the presence of proteins in conditioned media, where the most important proteins are probably those most abundantly secreted, because no reabsorbing or integrating into the sperm membrane could have occurred. Proteins found uniquely in the caput may be important in influencing specific maturational changes in spermatozoa as they transit the duct, whereas cauda-specific proteins may be important for the storage of spermatozoa. Such proteins may also be involved in regulating the structural and functional integrity of the epididymis itself.

Our results are consistent with the findings of Reyes-Moreno et al (2002); however, no protein identification was performed in our study. Reyes-Moreno et al (2002) have characterized 5 bovine proteins, secreted in cauda epididymal plasma, which could play a role in sperm protection in vivo. One protein identified as the beta-adrenergic receptor kinase 2 probably corresponds to protein 14 in the present study. Protein spots 10 and 11 (of about 48 kd) may correspond to the antithrombin-III and the fibrinogen gamma-B chain found by Reyes-Moreno et al (2002). In our study, these proteins were secreted in both regions of the epididymis. The beta chain (36-kd spot) of clusterin, which corresponds to protein 25 in our study, was secreted only by cauda epididymal epithelial cells in the presence of androgens. In vivo studies in the bull have shown similar regional secretions of the beta chain of clusterin (Howes et al, 1998).

It is concluded that androgens are involved in the regulation of protein synthesis by the epididymis. Identification and localization of these proteins on spermatozoa could provide useful information as to how spermatozoa can survive and preserve their metabolic quiescent state in the epididymis. To fully understand the regulation that occurs in the epididymis, the combination of molecular interactions between the sperm surface and the epididymal epithelium, ionic composition, and physiological conditions of the epididymal plasma must also be taken into account.

	Acknowledgments

 
The authors wish to thank J. Mestach and G. Spaepen for technical assistance.

	Footnotes

 
Supported by the Ministry of Agriculture of the Belgian Government, grant S6012, and by the Flemish Cattle Breeding Association VRV. This research was also possible by the Bilateral Collaboration Flanders-Quebec BIL 97/174 B 2298.

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