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a Dairy Breeding Research Center, Department of Dairy and Animal Science, The Pennsylvania State University, University Park, Pennsylvania 16802
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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OPN is a highly acidic glycoprotein that was initially isolated from the mineralized matrix of bovine diaphysial bone [3]. Subsequently it was detected in several tissues and fluids, including non-osteogenic cells. Distribution of OPN mRNA has been reported for bovine bone cells, tendon cells, cartilage, fetal skin, brain, kidney, ovary, uterus, and other tissues [4–9]. OPN has also been identified in a variety of fluids, including urine [10–13], bile [14], and bovine milk [15].
Brown et al. [14] studied the expression of OPN in normal adult human tissues, using in situ hybridization, immunohistochemistry, and Northern analysis. They showed that OPN was present on the luminal surfaces of epithelial cells of the gastrointestinal tract, gall bladder, pancreas, urinary and reproductive tracts, lung, bronchi, mammary and salivary glands, and sweat ducts. In general, Brown et al. [14] found that OPN accumulated on surfaces of epithelia bordering the luminal compartment.
Of interest to the present study was the observation of OPN in epithelial cells of the human prostate and in the epithelium of the rete testes [14]. More recently, Siiteri et al. [16] reported the presence of OPN mRNA in rat testis and epididymis and the detection of OPN in testis homogenates, epididymal fluid, and epididymal sperm extracts using Western blot analysis. Using reverse transcriptase-polymerase chain reaction, Siiteri et al. [16] also localized OPN on Sertoli cells and seminiferous epithelium. Indirect immunofluorescence of both fixed and unfixed sperm showed OPN immunoreactivity in the sperm tail and led Siiteri et al. [16] to conclude that OPN was a cell surface molecule.
The results of Siiteri et al. [16] led us to hypothesize that OPN in the bull reproductive tract would be found in the testis and epididymis and associated with spermatozoa. Because SP contains secretions of the accessory sex glands, epididymides, and testes, it also was of interest to determine the distribution of OPN in the male reproductive tract fluids. The objectives of this study were to localize OPN in tissues and fluids of the bull reproductive tract and to determine whether OPN associates with sperm.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Semen was collected using an artificial vagina after adequate sexual stimulation of the bull to maximize sperm harvest. Each ejaculate was centrifuged at 600 x g for 10 min at room temperature (RT), and the supernatant SP was recovered and recentrifuged at 10 000 x g for 1 h at 4°C. Plasma (BP) was harvested from blood recovered from the bull tail vein in a 10-ml syringe containing 2 ml of 2% EDTA in saline. Blood serum (BS) was obtained from whole blood after clotting. Accessory sex gland fluid (AGF) and cauda epididymal fluid (CEF) were obtained from bulls that were surgically equipped with indwelling catheters in the vasa deferentia [17]. Cauda epididymal sperm and CEF were collected from indwelling catheters in the vasa deferentia following ejaculation, while AGF was collected from the penis with an artificial vagina. Both the epididymal effluent and AGF were centrifuged at RT at 1000 x g for 10 min. The CEF supernatant was further centrifuged (10 000 x g for 1 h at 4°C) to remove any remaining sperm. After excision of the seminal vesicles from the slaughtered animal, seminal vesicle fluid (SVF) was obtained by massaging the gland and collecting the fluid in a plastic container. Ampullary fluid (AMF) was collected after slaughter by flushing the ampulla with 10 ml of PBS. After recovery, SVF and AMF were snap frozen in liquid nitrogen for transport to the laboratory. The samples were then thawed, centrifuged at 10 000 x g for 1 h at 4°C, and stored at -70°C until needed. Urine was obtained at time of slaughter by aspiration from the bladder, or by collection from live animals, and was frozen in liquid nitrogen for transport and storage. After thawing, urine was centrifuged at 10 000 x g for 1 h at 4°C, and the supernatant was collected and concentrated (1:10) using an Amicon (W.R. Grace&Co.-Conn., Beverly, MA) 10-kDa membrane.
All samples were then dialyzed against PBS (pH 7.4) containing 0.05 M EDTA, 0.2% sodium azide and stored at -70°C until needed. Protein from fluids of the bull reproductive tract were precipitated with cold (-20°C) 95% ethanol, washed, dried, and resuspended in PBS. Samples were stored at -70°C until needed. Protein concentration was determined using ovalbumin as standard [18].
Sperm Membrane Isolation
Cauda epididymal sperm were obtained from bulls surgically equipped with indwelling catheters in the vasa deferentia [17]. After sperm concentration and motility of each ejaculate were evaluated, each sample was centrifuged for 10 min at 600 x g at RT. The CEF was removed and the sperm pellet was washed twice in sterile PBS. Ejaculated sperm were obtained after semen centrifugation (600 x g for 10 min) and washed as described for cauda samples. Sperm membrane solubilization was performed as described by Jones [19]. Sperm were incubated in Jones's solubilization buffer (0.4% sodium deoxycholate, 0.26 M sucrose, 10 mM Tris, pH 8.5) at a concentration of 2.5 x 108 sperm/ml for 1 h at 4°C. The mixture was centrifuged at 10 000 x g for 30 min at 4°C. The supernatant containing the sperm membrane proteins was dialyzed three times in PBS at 4°C. Sperm membrane extracts were then concentrated using the Centricon10 microconcentrator (Amicon) by centrifugation at 1000 x g for approximately 1 h, and protein concentration was determined [18].
Protein Extraction from Tissues of the MaleReproductive Tract
Male reproductive tract tissues were obtained from bulls at slaughter. Tissues were excised, rinsed in PBS (pH 7.4), and immediately frozen in liquid nitrogen for transport to the laboratory and storage at -70°C. Protein extraction from accessory sex glands, testis, and epididymis (caput, corpus, cauda) was performed using TRI Reagent (Sigma Chemical Co., St. Louis, MO) according to the protocol outlined in Sigma Bio Sciences Technical Bulletin MB-205. Minor modifications in the final washing procedure were as follows. The protein pellet was dried and dissolved in 1% SDS solution. Insoluble material was removed by centrifugation at 10 000 x g for 10 min at 4°C. The supernatant was transferred to a new tube, and the protein concentration was determined [18]. Tissue proteins were then separated by one-dimensional (1D) SDS-PAGE, transferred to nitrocellulose, and analyzed using enhanced chemiluminescence (ECL) Western blot technique.
Antibody Production and Western Blot Analysis
Polyclonal antibodies against purified bovine seminal plasma OPN (55 kDa) were generated in New Zealand White male rabbits. OPN was recovered and electroeluted from spots (55 kDa, 4.5 pI) excised from 2D SDS-PAGE gels as previously described [2]. Electroeluted OPN (5 µg) was thoroughly mixed with Freund's complete adjuvant (1:1), and a total of 1 ml was injected s.c. at 10 sites in the scapular region. The rabbit was boosted twice, at 2-wk intervals, with purified OPN (5 µg total) mixed with Freund's incomplete adjuvant (1:1). The rabbit was bled 2 wk after the second boost by marginal ear vein, and serum was obtained by centrifugation at 1000 x g for 30 min at RT. Polyclonal antibodies generated against the N-terminus (LF123) and C-terminus (LF124) of human recombinant OPN were kindly provided by Dr. L.W. Fisher at the Bone Research Branch, NIDR-NIH, Bethesda, MD [20].
The ECL Western blotting protocol (Amersham International plc, Buckinghamshire, England) was used for localization of OPN in the male reproductive tract tissue extracts. Proteins were separated by 1D (50 µg) and 2D (500 µg) SDS-PAGE and transferred to nitrocellulose paper (Trans-Blot Transfer medium; Bio-Rad, Hercules, CA) using a Milliblot semi-dry transfer system (Millipore Corp., Bedford, MA) at 25 V for 30 min. Nitrocellulose blots were stained with Ponceau S (0.5% Ponceau S, 1% acetic acid in water) to assess the quality of the transfer and then destained in water. Blots were blocked with PBS solution containing 0.1% Tween 20 and 5% BSA overnight at 4°C. After overnight incubation in primary antibody (diluted 1:1000 in PBS, 0.1% Tween 20, 1% BSA), blots were washed for 30 min in PBS-Tween 20 and 1% normal goat serum. Then, after incubation for 2 h with peroxidase-conjugated goat anti-rabbit IgG (diluted 1:10 000 in PBS, 0.1% Tween 20, 1% BSA), blots were washed three times with PBS-0.1% Tween 20 (15 min each); this was followed by a final wash of 5 min in PBS. Companion blots were incubated with rabbit preimmune sera (NRS) during the primary antibody incubation as a negative control. Blots were then incubated in a 1:1 dilution of ECL detection reagents, covered with plastic wrap, and placed on a film cassette. Membranes were exposed to Fuji medical x-ray film (Fuji Ltd., Tokyo, Japan) for 3–15 sec and developed immediately. The dried film was then scanned using a Bio-Rad densitometer scanner (Model GS-670) and analyzed using the manufacturer's recommendations (Molecular Analyst; Bio-Rad Image Analysis Software).
Immunohistochemistry of Tissues from the Male Reproductive Tract
Tissues were obtained at time of slaughter, rinsed with PBS, frozen in liquid nitrogen, and placed at -70°C until needed. Tissues were cut into 0.5-cm cubes and were prepared for fixation while frozen. Tissues were placed in Histo Prep tissue baskets (Fisher Scientific, Pittsburgh, PA; cat. #15–182–218) and dropped in PBS-4% formaldehyde at 4°C for 2.5 h. Samples were rinsed in 70% ethanol (three 20-min rinses), then in 95% ethanol (three 20-min rinses), and finally in 100% ethanol (three 20-min rinses). Samples were then rinsed three times in xylene, transferred to fresh xylene for 10 min, and added to paraffin vials at 60°C for 2.5 h. Finally, samples were embedded, cooled, and stored at 4°C overnight.
Tissue sections were made on a microtome (5 µm), placed on gelatin-coated slides, air dried, and stored at 40°C for 1 h. A set of the slides was stained with hematoxylin for examination of the quality of the tissue, and the remaining slides were stored at 4°C until needed.
For antigen detection, slides were warmed at 42°C for 30 min, and tissue was rehydrated through a series of xylene and graded alcohols. Tissue slides were preincubated in 5% BSA, PBS-1% Tween 20 for 2 h at RT to block nonspecific reactions. Primary antibody (anti-55 kDa, LF123, or LF124) was diluted 1:100, and sections were incubated overnight at 4°C. A set of slides was incubated with preimmune serum as a control. The next morning, slides were washed twice with PBS, 1% BSA, 0.1% Tween 20 at RT.
Secondary fluorescein isothiocyanate-labeled anti-rabbit IgG (whole molecule adsorbed with human IgG; Sigma Immunochemicals, Sigma Chemical Co.) antibody was prepared as follows. The antibody was diluted to its working dilution (1:200) with PBS, 0.1% Tween 20, 1% BSA. Acetone-precipitated seminal vesicle protein powder (10 mg/ml) was added per 10 ml of the secondary antibody-containing PBS solution, and the mixture was incubated for 30 min at 37°C. The antibody was then centrifuged at 3000 x g for 20 min; the supernatant was recovered and filtered through a 0.22-µm membrane filter, and the filtrate was used for immunostaining. The secondary antibody was applied to the slides and incubated for 60 min at RT, after which slides were washed twice for 15 min with PBS, 0.1% Tween 20, 1% BSA. Tissue was counterstained with a 0.05% Evans Blue (Chroma-gesellschaft Schmid&Co., Roboz Surgical Instrument Co., Washington, DC) in PBS for 10 min and mounted using SlowFade (Molecular Probes, Eugene, OR) to reduce fluorescence fading. Slides were analyzed using an Olympus BHS microscope equipped with a reflected light fluorescence attachment (BH2-RFC) and an Olympus SC35 Camera (Olympus Optical Co., Tokyo, Japan).
Immunohistochemistry of Sperm
Cauda and ejaculated sperm were obtained as described above. Ampullary sperm were obtained by flushing the ampulla with 10 ml of PBS. After centrifugation, sperm were washed twice in PBS (1:10) and smeared on a gelatin-coated slide. Immunohistochemistry of sperm was performed as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Western blot analyses of fluids from the male reproductive tract separated by 1D SDS-PAGE showed that polyclonal antiserum raised against bovine seminal plasma OPN recognized a 55-kDa protein in SP, AGF, SVF, and urine (Fig. 1). ECL Western blots of all fluids probed with our anti-55 kDa detected two other protein bands at 45 kDa and ~14 kDa along with the 55-kDa protein. Although slight reactions were seen in lanes for BP and BS between 55 and 66 kDa, similar nonspecific reactions were seen in these lanes for blots of the preimmune control. The 55-kDa protein band and two additional proteins at 45 kDa and 14 kDa also were recognized by ECL Western blot analysis of 1D SDS-PAGE of SP, AGF, and urine samples probed with LF123 and LF124 antibodies (Fig. 2). Although the LF124 antibody recognized all three molecular weight forms of OPN, the LF123 antibody to the N-terminus of human recombinant OPN recognized only the 45-kDa and 55-kDa forms. Interestingly, the LF123 and LF124 antibodies to human OPN peptides recognized additional bands in BP.
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The findings that male reproductive fluids contained OPN were further confirmed by 2D SDS-PAGE and ECL Western blot analysis of SP, AGF, and SVF (data not shown). In addition, samples of AMF fluid were subjected to 2D SDS-PAGE analysis and ECL Western blot detection for OPN. In Western blots of AMF probed with antiserum against bovine seminal plasma OPN, 55-kDa, 45-kDa, and 14-kDa protein spots were detected (Fig. 3). A 70-kDa band was detected in some 2D SDS-PAGE Western blots, although this band appeared to be artifactual, as control samples probed with preimmune serum showed the same pattern.
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Although OPN was detected in 1D SDS-PAGE analysis of urine samples obtained from the bladder (Fig. 1), protein concentrations were found to average 46.0 µg·ml-1, which was about 10 times the concentration detected in urine samples obtained from live animals (4.39 µg·ml-1). Nevertheless, the presence of OPN in urine from live animals was confirmed by 1D SDS-PAGE ECL Western blot analysis using antiserum for seminal plasma OPN (Fig. 4).
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OPN in Protein Extracts of the Male Reproductive Tract
Western blot analysis of proteins extracted from the male reproductive tract tissues localized OPN in the seminal vesicle and ampulla (Fig. 5). In addition to the characteristic 55-, 45-, and 14-kDa OPN bands seen in SP, AGF, and SVF (Fig. 1), an additional band was seen in tissue extracts of ampulla, at ~32 kDa (Fig. 5). This 32-kDa band was also seen in SP on this blot, suggesting bull-to-bull variation in profiles or possible degradation of higher molecular weight forms during the extraction procedure. Two prominent bands were detected at ~12 kDa and ~27 kDa in tissue extracts, but not in SP samples using both OPN-specific antibodies and control sera. Although several different approaches were attempted in order to block binding to these bands, we were unable to eliminate the signal. This nonspecific binding occurred only on Western blot analyses of proteins extracted from tissues.
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Immunofluorescence Analysis of the Male Reproductive Tract and Sperm
Results of immunofluorescence analyses of the male reproductive tract paralleled results for tissue extracts and fluids, indicating that bovine OPN is secreted by the ampulla and seminal vesicle. Tissue sections of the testis, epididymis (caput, corpus, cauda), vas deferens, prostate, and bulbourethral gland were negative when reacted with antibodies against bovine seminal plasma OPN, LF123, LF124, and with NRS. When reacted with OPN antiserum, sections from the ampulla and seminal vesicle (Fig. 6) showed green fluorescence in the epithelia and the luminal surface that was not present when tissues were reacted with preimmune serum. Immunofluorescence of ejaculated ampullar and cauda sperm was negative for control and all OPN antisera, but positive for anti-bovine sperm membrane antibodies. Sperm preincubated in 0.1% Triton X-100 to permeabilize the membrane also were negative when probed with OPN antisera.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Results on the presence of OPN in male reproductive tissues from different species are inconsistent. Because we were unable to detect OPN in testicular and epididymal tissues and CEF using three different OPN antisera, we conclude that, in the bull, OPN in SP originates from the ampulla and seminal vesicles. This conclusion is consistent with the results of Yoon et al. [5] for the rat and of Craig and Denhardt [9] for the mouse; these investigators were unable to detect OPN mRNA in testis using Northern blot analysis. However, Siiteri et al. [16] recently identified OPN mRNA and OPN in the rat testis and epididymis, and Western blot analyses with anti-rat OPN antibody showed OPN immunoreactivity at 25–32 kDa, 53 kDa, 69 kDa, and 95 kDa in rat testis and epididymal tissue homogenates.
Several other explanations are possible for our inability to detect OPN in the testis and epididymis of the bull. It has been suggested that OPN function is altered by posttranslational modifications in various tissues [14]. It is possible that posttranslational modifications may have compromised the ability of the OPN antibody to detect OPN in these tissues. In this study three different antisera were used to detect OPN in the bull reproductive tract. However, the possibility that all three antisera would fail to detect OPN seems unlikely. Because antisera LF123 and LF124 were raised against the polypeptide backbone of human recombinant OPN, one would predict that at least one of these antisera would detect alternate forms of OPN in different tissues. Given the alternatives and available information, the most plausible explanation at the present time is that species differences exist for the distribution of OPN in male reproductive tissues.
The protein pattern seen in 2D SDS-PAGE gels of urine samples was similar to that for SP proteins. The unusually high protein concentrations detected in urine samples drawn from the bladder at slaughter may have resulted from retrograde ejaculation into the bladder, which could explain the presence of OPN and the similarity of the protein pattern with that of SP. In the bull, the colliculus seminalis swells to block the urethral orifice, and the muscles of the urethra contract so no urine passes at ejaculation [21]. At slaughter, when urine was collected from the bladder, it is possible that seminal fluid may have entered the bladder by backflow. To address this concern, urine samples were also taken from live animals. Although protein concentrations of urine samples taken from live animals were low, OPN was detected. However, the low protein concentrations found in these samples do not support the hypothesis that OPN in SP is a product of urine infiltration into semen. Moreover, this result confirms previous findings of detection of OPN in urine [11–13].
The present studies on the localization of OPN do not support the hypothesis that OPN binds to bovine sperm. Efforts to detect OPN in solubilized membrane extracts of epididymal and ejaculated sperm were unsuccessful (data not shown). Likewise, efforts to detect OPN by immunocytochemistry in epididymal, ampullar, and ejaculated sperm from bulls of differing fertilities also were unsuccessful (data not shown). These results are in contrast with those reported by Siiteri et al. [16], who localized OPN in the rat sperm tail using indirect immunofluorescence. It is possible that OPN is loosely associated with sperm and that OPN was stripped from sperm during the washing procedure. However, this possibility is inconsistent with the conclusion of Siiteri et al. [16] that OPN is an integral part of the sperm membrane. Alternatively, OPN detection may vary with the functional status of sperm, such as during capacitation or the acrosome reaction. This phenomenon has been previously described in relation to the localization of fibronectin on the sperm surface [22–24]. Studies on sperm surface fibronectin have shown that freshly collected sperm do not display fibronectin on their plasma membrane, whereas a significant number of sperm incubated overnight under capacitating conditions react with anti-fibronectin antibodies [25].
These findings, unfortunately, do not provide an apparent connection to explain the relationship between OPN in SP and male fertility. The relationship between OPN in SP and male fertility may be indirect. For example, Brown et al. [14] postulated that luminal OPN binds to integrin receptors located on the epithelial surface and may function to protect against bacterial infections and influence host defenses in inflammatory conditions. In this capacity, OPN could influence male fertility indirectly by protecting epithelial surfaces of the accessory sex glands from bacterial infections, thus having a positive influence on male fertility. Alternatively, OPN may modify characteristics of the sperm plasma membrane favoring increased fertility but its association with the membrane may only be transitory. Studies to define the role OPN plays in influencing male fertility are currently under way.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 814 863 0833; lwj{at}psu.edu
Accepted: September 25, 1998.
Received: April 28, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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